bluuebunny/crossref_metadata_2025 huggingface dataset:
Anemia Prevalence in a Population of Over One Thousand School Children in Northeastern Mexico
Authors: Escoboza Jose R Borbolla , Ugalde Patricia Argentina , Elizondo Luz Leticia , Patiño Beatriz Eugenia , Villela Luis M , Scott Sean P , Diaz Fernando Cristobal , Perfecto Yocanxochitl , Garcia Maria Isabel , Rocha Carlos , Kim Jina
Published: April 2020

Introduction: Anemia is a common finding in most developing countries. Of all populations, children are the ones who suffer most from anemia due to the long-term effects it can have on their growth and development. Large scale public programs to prevent anemia should always be based on epidemiological studies from significant sample sizes.

Patients and Methods: We included elementary school children from the state of Nuevo León, in northern Mexico (pop 4.2 million). We selected the children from eight public schools, six from Monterrey’s metropolitan area (pop 3.5 million) and two from the suburban area (pop. 0.5 million), mimicking the population distribution in our state. Three of these elementary schools give out school lunches consisting of milk and peanuts. Hemoglobin levels were obtained by using a Beckman-Coulter HMX® automated system (Miami, FL). We defined anemia as less than 12 g/dL of hemoglobin.

Results: After obtaining informed consent from the parents, we studied 1,137 children (550 males, 587 females), all of them between 6 and 12 years of age. Mean hemoglobin was 13.18 g/dL for females and 13.27 g/dL for males. We found 53 cases of anemia: 21 males and 32 females. Among the 53 anemic patients, 50 had hemoglobin between 11–12 g/dL, 1 between 10–11g/dL and 2 between 9–10 g/dL. We found significant differences in mean leukocyte numbers (7,200 vs 6,500/uL P=0.018); Mean Corpuscular Volume (82.9 vs 82.10 fL P=0.39); Mean Corpuscular Hemoglobin (29 vs 28.6 ug P=0.001), between the non-anemic and the anemic patients. Comparing the number of anemic children between schools with (28.7%) and without (71.3%) school lunches, the proportion of anemic children was significantly increased (26 vs 27 P=0.001) in schools that do not provide this school lunch.

Conclusions: Our results show a very accurate picture of the status of anemia in the urban and suburban areas of the state of Nuevo Leon in northeastern Mexico. Although one anemic child is one to many, our results show marked improvement when compared with our national means. Even though there are other factors that influence the absence of anemia in the different schools, the administration of the school lunch appears to have beneficial effects on anemia in our population.
Physicians and Patients Perception of a Problem as Ethically Relevant in Oncohematology: Only a Question of Perspective?
Authors: Giordano Giulio , Sacchini Dario , Farina Giuliana , Piano Silvia , de Paula Ignacio Carrasco , Storti Sergio
Published: April 2020

Ethical issues are connatural with the medical activity. Moral questions regarding some medical specialities as obstetrics or pre or perinatal medicine are well known. Nevertheless few is known about ethical problems perceived as remarkable from physicians and patients in onco-hematology. This is a retrospective monocentric descriptive study. Its purpose is to recognize, in oncohematology, what are the problems perceived as ethically relevant both from physicians and patients. Ethical issues regarding onco-hematologic patients were recognized consulting clinical diary of 100 patients treated in our institution. Patients have casually been selected in the temporal period from January 2004 to June of 2008. Ethical questions were listed in two groups: those perceived as remarkable for the physicians and those for the patients. Median age of the patients was 54.5 years (R 33–86). M/F was 55/45. Sixty-one patients were affected by chronic or indolent hemopathies. Sixty-six patients received 1st line therapy, instead 34 2nd line or superior. Seventy-five patients had primary school instruction, 22 secondary, 3 had got a degree. Eighty patients were assisted by a relative. Twenty-nine patients perceived a problem as ethically relevant (5 compliance to treatments; 5 refusal of treatment; 7 intolerance to hospitalization; 9 scarce trust in caregivers; 3 difficulty to understand the therapeutic plan). In 38 cases physicians perceived a problem as ethically relevant (8 over treatment; 7 type of treatment to adopt; 8 correct timing of therapy; 11 correct use of resources; 4 adequacy of treatment). Physicians perceived these problems mainly about patients requiring 2nd line treatment or further (28 cases out of 38). In 12 cases only patients perceived a problem, in 21 cases only physicians perceived a problem, while in 17 cases a problem was perceived as ethically relevant by physicians and patients simultaneously. In our study, in onco-hematology the main issue perceived by patients as ethically relevant is the scarce trust in caregivers, while for physicians it is the correct use of resources. Physicians perceive an ethical problem mainly considering patient candidate to 2nd line treatment or further. In 24% of cases, a problem perceived as ethically relevant by patients is underestimated by physicians. In this contest the possibility of an ethical consultation within a clinical counselling might create a convergence between physician and patient perspective.
Improving the Sensitivity and the Specificity of HLA-B27 Typing by Replacing Serological Tests with a TaqMan-PCR Assay
Authors: Weng Xiaoduan , Robin Louise , Audet Marie-lise , Hébert Linda , Lapointe Ginette , Major Micheline , Lemieux Bernard , Biron Guy , Bélanger Karl , Soulières Denis
Published: April 2020

HLA-B27 is a strong diagnostic biomarker as it is found in 90– 95% of ankylosing spondylitis. Routinely, the false-positive results generated by cross-reactivity of HLA-B27 monoclonal antibodies (mAbs) with some other type of HLA-B antigens are problematic. The present study aims at improving the sensitivity and specificity of typing for HLA-B27 by using a more accurate DNA assay. A real time sequence specific primer polymerase chain reaction (PCR-SSP) is performed by using MGB TaqMan oligoprobe and an ABI PRISM™ 7500 sequence detection system. The TaqMan PCR-SSP assay is compared with Immunophenotyping by flow cytometric antigen assay (BD HLA-B27-Kit, clone GS145.2). The results are finally confirmed by sequencing (ABI PRISM ® 3100). As summarised in the table, three methods are identical for clearly positive and negative results. There is 90% concordance for borderline negative results obtained by immunophenotyping with TaqMan PCR-SSC and/or sequencing. However, only 15.4% of borderline positives obtained by immunophenotyping are confirmed as true HLA-B27 positives by sequencing as well as TaqMan PCR-SSC. TaqMan PCR-SSP DNA assay completely correlates with sequencing (gold standard) with 100% PPV and NPV, compared to a PPV of 15% and a NPV of 98% for borderline results by immunophenotyping.

Conclusion: when compared with flow cytometric antigen assay, typing HLA-B27 by TaqMan PCR-SSP clearly demonstrates an advantage to better establish the genotype of the patient. Specifically, there is a marked difference for ambitious positive results from immunophenotyping. Moreover, compared to immunophenotyping, there is no need of fresh samples, can test a larger number of samples concomitantly, and reduces technical time as well as cost.

N = 52 Immunophenotyping TaqMan PCR-SSP Sequencing Immuno vs Sequencing TaqMan PCR-SSP vs Sequencing Positive 11 11 11 100% 100% Negative 17 17 17 100% 100% Positive (borderline by Immunophenotyping) 13 2 2 15% 100% Negative (borderline ) by Immunophenotyping) 10 9 9 98% 100%
A Mitochondrially Targeted Nitroxide JP4-039 Protects and Mitigates against Total Body Irradiation Induced Hematopoietic Syndrome
Authors: Epperly Michael W , Pierce Joshua G , Dixon Tracy , Franicola Darcy , Nie Suhua , Zhang Xichen , Vlasova Irina I , Wipf Peter , Kagan Valerian , Greenberger Joel
Published: April 2020

Nitroxides are efficient scavengers of free radicals and have been proposed for use as radioprotective agents. To circumvent the need for high concentrations for in vivo effects, and to target drugs to the mitochondria to prevent ionizing irradiation-induced apoptosis, we tested a family of compounds based on combining a hemi-gramicidin linker to a nitroxide. One small molecule (JP4-039) produced a 32-fold increased mitochondrial nitroxide localization in 32D cl 3 cells in vitro after one hour incubation at 37°C compared to the unlinked nitroxide tempo at 10uM. The cells were lyzed by rapidly freezing then thawing, followed by centrifugation to yield the nuclear, cytoplasmic, and mitochondrial fractions. EPR was used to quantitate nitroxide in each JP4-039 or tempo treated cell fraction. There was a 32-fold increase in the nitroxide signal for JP4-039 accumulation in the mitochondria compared to the nitroxide tempo. Irradiation survival curves were performed by incubating 32D cl 3 murine hematopoietic progenitor cells in JP4-039 or tempo (1 μM) for one hour before irradiation or by addition immediately following irradiation. The cells were irradiated to doses ranging from 0 to 8 Gy, suspended in methylcellulose-containing media, incubated for seven days at 37°C and colonies of greater than 50 cells were scored. The data was analyzed using linear quadratic and single-hit, multi-target models. Incubation of 32D cl 3 cells in JP4-039 for one hour prior to irradiation resulted in a significant increase in Do to 2.25 ± 0.11 Gy compared to 1.19 ± 0.13 Gy for 32D cl 3 cells alone or 1.32 ± 0.09 Gy for 32D cl 3 cells incubated in tempo (p=0.0042 or 0.0073, respectively). Similar results were achieved giving JP4-039 after irradiation with a Do of 1.97 ± 0.01 Gy (p < 0.0001). To test effectiveness in vivo, C57BL/6NHsd female mice were injected intraperitoneally with JP4-039 or tempo (10 mg/kg) 10 minutes before (or four hours after) total body irradiation to the LD 75/30 dose of 9.75 Gy. Mice were monitored and those moribund from the hematopoietic syndrome were sacrificed. Mice injected with 10 mg/kg JP4-039 before irradiation had a significant increase in survival of 60% compared to 28% for control irradiated mice (p = 0.0005) or 44% for tempo treated mice (p = 0.2252). JP4-039 given after irradiation had a survival of 53% at 30 days after irradiation (p = 0.0474). Thus, JP4-039, a mitochondrial targeted nitroxide, is an effective radioprotector, and mitigator.
Extracellular HMGB1 Enhances Proliferation, Differentiation and Migration of Human CD34+ Hematopoietic Stem Cells in Vitro
Authors: Wang Xingbing , Chen Xin , Ren Weihua , Xu Xiucai , Song Kaidi , Liu Huilan , Sun Zimin , Huang Shiang
Published: April 2020

High-mobility group box 1 protein (HMGB1) is a chromatin protein and acts as a cytokine involved in inflammation, cell proliferation, differentiation, migration and stem cell recruitment. So far, little is known about its effect on hematopoietic stem cells (HSCs). In this study, we investigated whether receptors for HMGB1 are expressed on human CD34+ HSCs, and whether HMGB1 could affect HSCs proliferation, differentiation and migration in vitro. As examined by FACS analysis and RT-PCR, cord blood CD34+ HSCs express the HMGB1 receptors RAGE (receptor for advanced glycation end products), TLR2 (Toll-like receptor 2) and TLR4. To study the effects of HMGB1 on CD34+ HSCs proliferation and differentiation, freshly isolated cord blood CD34+ HSCs were cultured for 7 days in medium alone or in the presence of HMGB1. Flow cytometric analysis showed that HMGB1 (50ng/ml) can induce the differentiation of CD34+ HSCs along the granulo-monocytic (CD14+, CD13+) lineage and erythropoiesis (CD71+). In contrast, HMGB1 did not induce the expression of CD41, a marker for megakaryocyte lineage. The numbers of cells cultured in the presence of HMGB1 were always increased in comparison with controls. Furthermore, higher numbers of granulomonocytic progenitors (CFU-GM), erythroid progenitors (BFU-E), and CFU-MIX were confirmed by CFC assays in a HMGB1 dose dependent manner after 14-day culture. The results suggest that HMGB1 enhances CD34+ HSCs proliferation and differentiation. We next assessed the effects of HMGB1 on HSCs migration by chemotaxis assay using Boyden chambers. HMGB1 dose-dependently increased the chemotactic migration of CD34+ HSCs. A neutralizing anti-RAGE antibody significantly blocked the HMGB1-induced migration of HSCs, whereas neutralizing TLR2 and TLR4 antibodies did not significantly influence HMGB1-stimulated HSCs migration, suggesting that the migratory effect of HMGB1 on human HSCs is predominantly mediated by RAGE. In summary, our results provide the first report of HMGB1 receptors expression profile of human cord blood CD34+ HSCs and demonstrate that HMGB1 can increase the proliferation and migration of HSCs and directly induce of HSCs along myeloid differentiation and erythropoiesis in vitro. Further studies will be needed to clarify the mechanism of HMGB1 activation and the physiological function of HMGB1 in HSCs in vivo.
Expression of Functional Toll-Like Receptor 2 on Mouse Embryonic Stem Cells
Authors: Taylor Tammi , Derbigny Wilbert , Kim Young-June , Broxmeyer Hal E.
Published: April 2020

Embryonic stem (ES) cells have the capacity to produce all cell types of the body. Understanding murine ES (mES) cell proliferation, survival, differentiation, and self renewal will enhance knowledge of developmental biology and essential use of ES cells. Recently, Toll Like Receptor (TLR) activation has been shown by others to play a role in influencing the differentiation of hematopoietic stem cells. Previous studies have also shown that TLR activation prevents mesenchymal stem cell differentiation into adipocytes, chondrocytes, and osteocytes and plays a role in bone repair. We hypothesized that certain TLR’s would be expressed on mES cells and that the ligands for these expressed TLR’s would induce functional activity in the mESC’s. Therefore, we wanted to determine if TLRs are expressed on mES cells and if so, are they functional. Three different mES cell lines (R1, CGR8, and E14) were used to determine if TLRs are expressed at the mRNA level using primers for murine TLR1-9 mRNA. We found that TLR’s 1, 2, 3, 6, and 9 were expressed at the mRNA level, but TLR’s 4, 5, 7, and 8 were not. Based on the availability of antibodies to TLR’s, and using flow cytometry, we found expression of TLR2 but not TLR 4 on the surface of all three mES cell lines. TLR ligands were used to treat mES cells in the presence of leukemia inhibitory factor (LIF) for an hour. Activation of TLR2 by its ligand Pam3Cys, a synthetic tri-acyl lipoprotein, on mES cells induced NF-κβ nuclear translocation when compared to ES cells not stimulated with TLR ligands. LPS, the ligand for TLR4 did not induce NF-κβ nuclear translocation on ES cells, consistent with lack of cell surface expression of TLR4 on mES cells. TLR expression and TLR ligand interaction were not associated with changes in the morphology of the mES cells or expression of Oct-4, SSEA-1, KLF-4, or Sox-2, markers for maintenance of the undifferentiated state of mES cells. This suggests that the cells remain in an undifferentiated state even after TLR activation by Pam3Cys in the presence of LIF. Thus our study has identified functionally active TLR2 on the surface of mES cells, information that may be of use to further defining a role for TLR’s on ES cells, and for manipulation of other ES cell functions.
Study on the Mechanism of ‘ei’formation in the Bone Marrow Smears of the Patients with BMMNC-Coombs Test(+) Hematocytopenia
Authors: Fu Rong , Wang Yi hao , Shao Zonghong , Chen Jin , Li Lijuan , Liu Hui , Wang Huaquan , Xing Limin , Liu Hong
Published: April 2020

Objective To explore the mechanism of ‘erythroblastic island(EI)’ formation in the bone marrow of the patients with BMMNC-COOMBS( {)hematocytopenia.

Methods The category of BMMNC (hematopoietic stem cells, nucleated erythrocytes, granulocytes) -Ab in 48 patients with BMMNC-COOMBS( {) hematocytopenia were detected with FACS. The autologous antibody in the“EI” of these cases were explored with immuonhistofluorescence(IF). Clinical and other laboratory characteristics of these cases were also analyzed retrospectively.

Results Mouse anti-hIgG could be detected in the ‘EI’on the BM smear of 14 cases(29.17%), autologous antibody mainly deposited at the edge/membranes between macrophage and erythroblasts rather than in the cell plasma. Positive reaction were seen in all the cases with BMMNC-GlycoA IgG(+). The initial manifestation of the 14 patients was anemia. The percentage of reticulocyte (1.98%) and the serum level of IBIL of these cases (9.39 mmol/l)(5–24.90 mmol/l) were elevated in the 14 patients; Hypercellularity with higher percentage of erythroid (44.14%) in BM was seen in most patients. These cases had good reponse to high dosage of IVIgG (HDIVIG) or/and Glucocorticoids. There were significant difference of these indices between the IF(+), the IF(−)group and control group.

Conclusion Macrophage was connected with erythroblasts through autologous IgG in the‘EI’s of some patients with BMMNC-Coombs test(+) Hemocytopenia. These ‘EI’s were the places where macrophages devoured and destroyed erythroblasts rather than the places where erythroid development and differentiation. The pathogenic mechanism of this kind of disease might be associated with macrophages phagocyting and destroying more BM hematopoietic cells.
Phenotypic Characterization of Human CD34+ Cells Selected from G-CSF Mobilized Leukopheresis Using Baxter’s Isolex™ 300i Magnetic Cell Selection System (v2.5)
Authors: Cohen Amy , Motlagh Delara , Amrani David
Published: April 2020

Baxter’s Isolex™ 300i Magnetic Cell Selection System (v2.5) is used to select CD34+ cells from G-CSF mobilized leukopheresis units. This study focused on evaluating the cellular composition of the post-Isolex product as well as producing a detailed characterization of the CD34+ cells. A multi-color flow cytometry assay was developed to characterize the Isolex product with a detailed focus on the CD34+ cells. A hematopoietic colony-forming assay was used to determine the clonal potential of the selected cells. Cell function was evaluated by measuring the migration potential of the CD34+ cells toward SDF-1. The post-Isolex cell suspension product contained an average of 96.44% ± 0.84% CD34+ cells. The majority of the remaining cells present were T and B cell lymphocytes with an average 1.74% ± 0.78% CD3 positive T-Cells and 1.61% ± 0.23% CD19 positive B-Cells. The platelets comprised 1.92% ± 0.92% of the product and 99% of this population were co-aggregated to the CD34+ cells. Non-CD34, immature white blood cells averaged less than 0.5% of the total post-Isolex population. A majority of the CD34+ stem cells are lineage-committed HPCs as determined by KDR-CD34+ (94.90% ± 1.99%) and CD38+CD34+ (93.87% ± 2.76%). Commitment to a myeloid cell line (CD33+CD34+) was determined to be 34.75% ± 23.10%. Migration potential of the stem cells toward SDF-1 as defined by dual positivity for CXCR4 and CD34 averaged 1.95% ± 2.49%. While the CXCR4 receptor positivity appears low, the migratory response of the CD34+ cells toward SDF-1 was robust. An examination of VEGF populations in the post-Isolex product averaged 0.32% ± 0.17% for VEGF R2 (KDR) and 34.25% ± 12.74% for VEGF R1. Several adhesion markers were assayed and the positivity was equal to or less than 1% for CD146, CD144, CD73, and CD29. Other adhesion markers, such as CD105 (endoglin) averaged 34.04% ± 20.64% and CD99 averaged 66.33% ± 13.25%. CD117, which binds to the receptor for stem cell factor (SCF), averaged 69.63% ± 6.38%. CD133, which is considered an early HPC marker, averaged 73.90% ± 5.97%. CD90, which is useful for defining a population of highly proliferative cells capable of long-term culture, averaged 60.50% ± 8.50%. The EPC phenotypic profile combination, KDR+CD34+CD133+, averaged 0.30% ± 0.22% in our post-Isolex population. In the hematopoietic colony-forming assay, the Isolex-selected CD34+ cells were able to produce colonies with an efficiency of 20% with CFU-GM forming a majority of the colonies. By utilizing multi-color flow cytometry, we were able to account for the cell populations found in the Isolex product and to further characterize the CD34+ selected cell population. The hematopoietic colony-forming assay gave insight into the efficiency and growth potential of the CD34+ selected cells. The functional migration assay determined that the CD34+ selected cells exhibited a robust migratory response toward SDF-1.
A Hypoxic Niche in the Mouse Bone Marrow Diminishes Proliferation and Differentiation of Hematopoietic Stem Cells
Authors: Eliasson Pernilla M , Jönsson Jan-Ingvar
Published: April 2020

In the bone marrow hematopoietic stem cells (HSCs) reside in specialized niches in close contact with stromal cells and endosteal osteoblasts. It is thought that this environment is hypoxic in nature, where HSCs are maintained in a quiescent state to prevent their depletion. Hypoxia stabilizes the transcription factor HIF-1α which triggers angiogenesis as well as genes slowering the cell cycle, promoting cell survival, and leading to a decrease in cellular metabolism. In this study, hypoxic effects of the maintenance of Lin−Sca1+c-kit+* (LSK) cells derived from mouse bone marrow and the involvement of the transcription factor hypoxia inducible factor 1 α (HIF-1α) were investigated. Hypoxic culture conditions led to an increase in numbers of primitive colony-forming progenitor cells and a preferential expansion of immature blast-like appearing cells. Concurrently, the immature c-kit Sca-1 phenotype was better maintained in hypoxia compared to ambient oxygen levels. Moreover, hypoxia decreased the proliferation of HSCs as measured by CFSE or PKH26 staining. This was confirmed by cell cycle analysis, and hypoxic cultivation decreased the percentage of cells in S-phase whereas cells in G0/G1 phase increased. Cells infected with a constitutively active form of HIF-1α showed the same pattern as cells cultured in hypoxia. To verify that the effect is HIF-1α mediated, we silenced HIF-1α in LSK cells with shRNA. The decrease in proliferation in hypoxic cultivation of cells infected with shRNA against HIF-1α was markedly diminished, indicating that HIF-1α play an important role in controlling proliferation of hematopoietic stem cells. These results suggest that a major function of hypoxia is to counteract proliferation and possibly differentiation, thereby sustaining maintenance. Furthermore, hypoxic culture conditions may have beneficial clinical implications for ex vivo purposes and may improve the yields of stem cells. In our ongoing-studies, we are investigating whether HIF-1α and hypoxia is an absolute prerequisite for the proper maintenance of HSCs in the bone marrow.
Hematopoietic Stem - and Progenitor Cells Are Differentially Mobilized in Response to Flt3-Ligand
Authors: de Kruijf Evert-Jan F. M. , van Pel Melissa , Fibbe Willem E.
Published: April 2020

Flt3 is a tyrosine kinase receptor expressed mainly on primitive hematopoietic cells and its ligand, Flt3-ligand (FL), is a slowly mobilizing agent in mice when administered for 5–10 days. This provides a time-frame to study the HPC and HSC migration kinetics in detail. BALB/c mice were injected for 3, 5, 7 and 10 days with FL (10 ug/day, intraperitoneally). Mobilization of hematopoietic stem- and progenitor cells was studied using colony-forming-unit granulocyte/monocyte (CFU-GM) and cobblestone-area-forming-cell (CAFC) assays. The radioprotective capacity of mobilized peripheral blood mononuclear cells (PBMC) was studied by transplantation of 1.5 × 106 FL-mobilized PBMNC into lethally irradiated (9.5 Gy) recipients. Hematopoietic progenitor cell mobilization was detected from day 3 onwards and prolonged administration of FL showed a steady increase in mobilized progenitor cells (CFU-GM; 23.1±20.1 [PBS], 219.0±213.7 [3 days FL, n=10], 1,007.8±742 [5 days FL, n=21], 3,526.6±1,406 [7 days FL, n=10] and 19,149±2,338 [10 days FL, n=10]). Administration of FL for 10 days lead to a 5.5-fold increase in CAFC-week 4, compared to FL administration for 5 days (0.69 vs 0.13 CAFC per 105 PBMC respectively) and a 5.0-fold increase in CAFC-week 5 (0.27 vs 0.05 CAFC per 105 PBMC respectively). No CAFC week 4/5 were found in PBMC obtained from PBS-injected control mice. Transplantation of 5-day FL-mobilized PBMC did not radioprotect lethally irradiated recipients (mean survival time (MST): 66 days; MST PBS: 13 days). In contrast, transplantation of PBMC obtained from a 10-day FL mobilization regime radioprotected 100% of the recipients (Table 1). These results indicate that HPC and HSC show different mobilization kinetics in response to FL, resulting in preferential mobilization of HPC at day 5, followed by HSC mobilization at day 10. We speculate that this difference in mobilization kinetics might be due to the selective recruitment of HPC and HSC from their respective bone marrow compartments.

Table 1. HPC and HSC frequencies in peripheral blood following Flt3-ligand-induced mobilization

CFU-GM per ml PB CAFC-day 28 per 105cells CAFC-day 35 per 105cells % Radioprotection day 150 * fold increase compared to 5 day FL 5 days Flt3-L 1,007 (n=21) 0.126 0.054 0 (n=10) 10 days Flt3-L 19,149 (n=10) (19.0)* 0.694 (5.5)* 0.272 (5.0)* 100 (n=10)
Hemodynamic Monitoring during Erythrocytapheresis in Sickle Cell Disease Patients Using Impedance Cardiography
Authors: Das Bibhuti B , Kemparajurs Samyuktha , Bertolone Salvatore , Kong Maiying , Raj Ashok B.
Published: April 2020

BACKGROUND: Erythrocytapheresis procedures are increasingly used in the treatment of severe acute complications of sickle cell disease (SCD). Some patients who undergo erythrocytapheresis experience dizziness during or after the procedure. There is no data in literature for hemodynamic monitoring of cardiac index (CI) and systemic vascular resistance index (SVRI) during erythrocytapheresis. We have previously shown that impedance cardiography (ICG) and invasive measures of CI by thermodilution techniques are significantly correlated in children.

OBJECTIVES: We wanted to evaluate changes in CI and SVRI using ICG and to correlate these with systolic blood pressure (SBP) measurements in SCD patients while undergoing erythrocytapheresis.

METHODS: Thirty one SCD patients who had 70 consecutive erythrocytapheresis procedures were included in this IRB approved study. ICG measurements (CI, SVRI, and blood pressure) were taken pre-erythrocytapheresis, 30 minutes after initiation of the procedure, one hour into erythrocytapheresis, and at the end of the procedure. ICG measurements were repeated in symptomatic patients before and after intervention with fluid administration.

RESULTS: We evaluated hemodynamic data on 31 SCD patients, average age 12.8±5.2 years and 56% are female. SBP decreased below the base line during 33 procedures and increased above the baseline during 37 procedures. Among patients who had a decrease in SBP, 28/33 (84%) had decreased CI. Among them 22/28 (78%) had increased SVRI. Among patients who had an increase in SBP, 5/37 (14%) had increased CI. Among them 4/5 (80%) also had increased SVRI. Using paired t test, the changes in CI (P <0.001) and SVRI (P=0.0002) comparing to baseline are statistically significant. CI is negatively correlated with change in SVRI (Pearson correlation coefficient −0.8).

CONCLUSIONS:In summary, the hemodynamic changes during erythrocytapheresis are probably multifactorial in origin and may include a combination of SVRI and CI changes. Confirmation of these findings may allow early intervention in some patients and minimize the risk of developing overt clinical symptoms during erythrocytapheresis.
Association of Pulmonary Hypertension with Sickle Cell Disease Subtypes
Authors: Cohen Alice J. , Tuckman-Vernon Chaim
Published: April 2020

Pulmonary hypertension (PH) is a common complication of sickle cell disease (SD) and a significant cause of morbidity and mortality. PH, measured by Doppler echocardiography and defined as a tricuspid regurgitant jet velocity (TRV) > 2.5 m per second (m/s), is hypothesized to be related to the chronic hemolytic anemia of SD, but causality is unproven. If so, the presence of hemoglobin C, which reduces hemolysis, would be expected to have a reduced likelihood of PH. This study reviewed the prevalence of PH in 3 categories of patients with SD: homozygous S (SS), sickle-beta thalassemia (SB), and SC.

Methods: Sickle cell disease patients registered at a state funded community comprehensive care adult sickle cell center were routinely screened for PH by Doppler echocardiography. The presence of PH, the incidence of a related complication, acute chest syndrome (ACS), and baseline hemoglobin (hgb) were reviewed.

Results: 16 patients with SC type, 30 with SS and 39 with SB disease underwent screening. The prevalence of PH, ACS and hgb are listed in the table below.

Conclusion: SC patients have PH and ACS similar to patients with SS and SB patients. These patients have higher baseline hemoglobin and may have hyperviscosity as a cause of PH and ACS as opposed to hemolytic anemia. Further study of PH and ACS in SC patients is warranted.

SC SS SB p value PH 6/16 (38%) 12/40 (40%) 11/39 (28%) p= NS ACS 7/16 (44%) 10/30 (33%) 19/39 (49%) p=NS PH + ACS 4/16 (25%) 5/30 (17%) 4/39 (10%) p=NS ACS in PH patients 4/6 (67%) 5/12 (42%) 4/11 (36%) p-=NS Hgb 10.8 7.89 8.57 p=0.000
Marijuana Use and Sickle Cell Disease
Authors: Gotlieb Vladimir K. , Oo Khine Z.
Published: April 2020

A 45-year old Jamaican man with sickle cell disease came to our clinic complaining of chronic left ankle ulcer for one year. He stated that when he was a young boy he had had several hospital admissions for sickle cell crisis, usually precipitated by physical activity or infection, once or twice per month. We were surprised to find out that he had never had a crisis since the age of fifteen. We doubted his diagnosis yet his hemoglobin electrophoresis revealed HbS was 94.7%, HbF was 0.6% and HbA2 was 4.7%. He denied taking any medication. However, he admitted smoking 4 to 5 cigarettes of marijuana a day for the past 30 years. Cannabis has been used as a medicine even before the Christian era in Asia, first in China then mainly in India. Its use was later spread to the West.

The general indications for marijuana were reported as severe nausea and vomiting, weight loss associated with debilitating illnesses, spasticity, pain syndrome, and glaucoma.

Numerous Phase I–III studies in 2000 subjects with exposure of Sativex by GW Pharmaceuticals from United Kingdom demonstrated that the patients attained good sleep quality, which may improve patients’ quality of life in disabling chronic pain syndromes.

We reviewed the literature on any report that cannabinoids could change the severity of sickle cell disease. We found the study from West Indies, which investigated the perception that marijuana use ameliorated the complications of sickle cell disease, in year 2000 and 2004. The study concluded that marijuana smoking is common in adults with sickle cell disease but its usage is unrelated to clinical severity of the disease.

We also found a report, from Central Middlesex Hospital in London, United Kingdom, which observed 86 young adults with HbSS, HbSC and HbSbetathalassemia disease, median age being 30 years.

Results of this study showed that 31 (36%) had used cannabis in the previous 12 months to relieve the symptoms associated with sickle cell disease. Symptoms related to sedation and mood effects were reported in 77% of patients. The main reasons for use of cannabis were to reduce pain in 52% and to induce relaxation or relieve anxiety and depression in 39%. (5) We wondered if any physicians had observed similar effects of marijuana in sickle cell disease patients in their practice. Could ‘Marijuana Use has beneficial effect on Sickle Cell Disease’ be an interesting topic for a broader clinical trial or a retrospective analysis?
Codanin-1, the Protein Encoded by the Gene Mutated in Congenital Dyserythropoietic Anemia Type I (CDAN1), Is Cell Cycle Regulated
Authors: Noy-Lotan Sharon , Dgany Orly , Lahmi Roxane , Marcoux Nathaly , Krasnov Tanya , Yissachar Nissan , Ginsberg Doron , Motro Benny , Resnitzky Peretz , Yaniv Isaac , Kupfer Gary , Tamary Hannah
Published: April 2020

Congenital dyserythropoietic anemia (CDA) type I is an inherited autosomal recessive macrocytic anemia associated with ineffective erythropoiesis and the development of secondary hemochromatosis. Distinct erythroid precursors with inter-nuclear chromatin bridges and spongy heterochromatin are pathognomonic for the disease. The mutated gene (CDAN1) encodes a ubiquitously expressed protein of unknown function, codanin-1. Based on the morphological features of CDA type I erythroblasts and the preliminary data on the Drosophila homolog, dlt, which was found to be required for cell survival and cell cycle progression, we investigated the location and the behavior of codanin-1 during the cell cycle. Using immunofluorescence and immune electron microscopy, we localized codanin-1 to the heterochromatin in interphase cells. During the cell cycle, high levels of codanin-1 were observed in S phase. At mitosis, codanin-1 underwent phosphorylation, which coincided with exclusion from condensed chromosomes. The proximal CDAN1 gene promoter region, never before characterized, was found to contain 5 putative E2F1 binding sites. E2F transcription factors are the main regulators of G1/S transition. Cotransfection of an E2F1 expression plasmid increased luciferase activity, confirming that E2F1 activates the transcription of CDAN1, and chromatin immunoprecipitation identified the codanin-1 promoter as a direct target of E2F1. Taken together, these data suggest that codanin-1 is a cell cycle-regulated protein active in S-phase. Based on the localization of codanin-1 to the heterochromatin and the spongy appearance of heterochromatin in CDA I, we suggest that codanin-1 may be involved in heterochromatin organization during DNA replication. This represents the first work towards understanding the function of the proteins involved in CDAs.
Hodgkin Disease and Nephrotic Syndrome, a Rare Paraneoplastic Complication in Children with Literature Review
Authors: Dsilva Sehba , Karayalcin Gungor , Singh Sharon
Published: April 2020

The association between Hodgkin Disease (HD) and paraneoplastic Nephrotic Syndrome is well documented in adults but is relatively uncommon in the pediatric population. We describe two children with HD who initially presented with Nephrotic Syndrome. Case 1: 12- year-old boy who presented with a two-week history of periorbital edema, proteinuria and hypertension and was diagnosed with Nephrotic Syndrome. The renal biopsy showed minimal change disease. Since the patient developed dyspnea, further work-up was done and he was found to have a large mediastinal mass. Core needle biopsy of the mass was consistent with HD, nodular sclerosing type. Case 2: 14-year-old boy diagnosed to have Nephrotic Syndrome with biopsy proven minimal change disease and was treated intermittently with steroids over 6 months. He subsequently developed dyspnea and was found to have a right paratracheal mass and excisinal biopsy was consistent with HD, nodular sclerosing type. Both these patients initially presented with Nephrotic Syndrome which completely resolved after the first course of chemotherapy for HD. Upon review of literature, there have been 23 pediatric cases of Nephrotic Syndrome associated with HD in patients ranging in age between 2–15 years. The renal biopsy was not done for 8 of these patients and the diagnosis was established clinically. Fifteen patients had biopsies, 9 had minimal change disease, 1 mesangial hypercellularity, 1 glomerulosclerosis, 1 undetermined histology and 3 were normal. In all cases, the Nephrotic Syndrome was refractory to steroids and resolved with treatment of the HD. Pathogenesis of the Nephrotic Syndrome associated with HD is not known, however there are speculations about T-cell involvement and production of lymphokines and humoral factors which may be responsible for the glomerular damage.
Caspofungin for the Treatment of Candidemia in patients with Hematological Malignancies
Authors: Fianchi Luana , Fanci Rosa , Candoni Anna , Caira Morena , Valentini Caterina Giovanna , Posteraro Brunella , Morselli Monica , Farina Giuliana , Mitra Maria Enza , Offidani Massimo , Sanguinetti Maurizio , Tosti Maria Elena , Nosari Annamaria , Viale Pierluigi , Leone Giuseppe , Pagano Livio
Published: April 2020

Objective: To evaluate the efficacy of Caspofungin in patients with hematological malignancies (HM) and candidemia

Design: The study was prospectively conducted in 11 Hematology Divisions in a tertiary care or university hospital setting.; neutropenic hematological patients with clinical evidence of infection and a positive blood culture for Candida were enrolled.

Patients and results: Between January 2005 and November 2007, 38 episodes of candidemia among patients with HM were registered. Among these, 24 eligible neutropenic patients, collected in 8/11 participating centers, were evaluated. All these patients had received chemotherapy for their underlying hematological diseases: acute myeloid leukemia (AML) in 11 patients (46%); non Hodgkin’s lymphoma (NHL) in 6 patients (25%); acute lymphoblastic leukemia (ALL) in 4 patients (16%); multiple myeloma (MM) in 2 patients (8%); chronic lymphoblastic leukemia (CLL) in 1 patient. In 11 patients (46%) the infection onset after a HSCT procedure (7 allogeneic and 4 autologous). Before the infection onset, all patients were neutropenic (ANC<0.5×109/l) for a median average of 12 days (range 2–41). Candidemia diagnosis was made after a median time of 3 days from the onset of fever. Candida isolated species were: C.albicans 46%, C. parapsilosis 17%, C. krusei 12.5%, C. tropicalis in 8%, C. lusitanae, C. guillermondii, C. dubliniensis, C. famata in 4% of patients each. Caspofungin at standard dosage was the first-line therapy in 17 pts (71%); in the other 7 pts, previous antifungal therapy (4 L-AmB, 1 itraconazole, 1 fluconazole, 1 voriconazole) was changed to Caspofungin at candidemia diagnosis after no more than 48 hours of treatment. The median duration of Caspofungin therapy was 12 days (range 3–26). No side effects were registered. A complete response to therapy of 58% was obtained. At 30 days following candidemia onset, 11 pts had died (41%), but the candidemia was responsible for the mortality in only 6 pts (overall attributable mortality 25%).

Conclusions: Our data confirm the efficacy of Caspofungin in the treatment of neutropenic pts with HM and concomitant candidemia as well as it was reported for non hematological subgroups. Caspofungin was well tolerated also in very compromised pts.
Do Deletions in 9p Reflect Prognosis in Adult Precursor B-Cell ALL? a Multi-Centre Study of 381 Patients
Authors: Nahi Hareth , Smedmyr Bengt , Hallböök Helene , Hagglund Hans
Published: April 2020

Despite the fact that the outcome among patients with acute lymphoblastic leukaemia (ALL) has improved, the majority of adult patients relapse and die of their disease. Besides age and white cell count at diagnosis, the cytogenetic abnormalities t(9,22)/BCR-ABL and t(4,11) are important prognostic markers and are often included in the treatment stratification of patients with adult ALL. Deletions in 9p are seen in about 9% of cases of adult ALL, but their prognostic impact has been controversial.

Methods: Cytogenetic data from 381 patients diagnosed with B-precursor ALL were reviewed. Chromosomal analysis was successful in 240 cases. Of these cases, 18 (8%) had abnormalities in 9p and they were compared with patients with normal karyotypes and patients with t(9;22)/BCR-ABL.

Results: Patients with abnormalities of chromosome 9 showed significantly shorter overall survival compared with patients with normal karyotypes. In fact, overall survival was similar to that in the poor prognosis t(9;22)/BCR-ABL-positive group.

Conclusions: The data suggest that chromosomal abnormalities involving 9p may have a significant negative impact on survival in adult B-precursor ALL.
Clinical Evaluation of a Custom Genome Wide 44K Oligoarray for Copy Number Changes in Acute Myeloid Leukemia
Authors: Patel Ankita , Wang Rui-Yu , Hixson Patricia , Li Jian , Milosavljevic Aleksandar , Kantarjian Hagop M , Cheung Sau w , Andreeff Michael
Published: April 2020

Oligonucleotide-based array CGH (aCGH) technique enables detection of submicroscopic copy number changes in cancer. This technique offers the flexibility to

increase robustness by selecting best performing probes maximize the gene coverage while excluding repetitive sequences through a combination of bioinformatics and computation and increased resolution that allows for detection of smaller region of change. We designed a high resolution (44,000 probes) custom array using an Agilent platform and specifically targeted ~500 genes implicated in leukemogenesis.

The disease gene regions have an average spatial resolution of ~1 oligo per 7.5 kb whereas the backbone regions have an average resolution of 1 oligo per 78 kb. A total of 13 samples from patients with AML have been studied to date. aCGH was performed in a blinded fashion and the results were compared to the results from routine cytogenetic analysis. A GAIN in copy number on the long arm of chromosome 8 at band 8q22.2 encompassing 2.05 Mb was observed in one of 7 cases with normal chromosome analysis. Of the remaining 6 cases, the results from aCGH were concordant with the abnormality detected by chromosome analysis in three although the size of the copy number change was determined with greater precision by aCGH. Additional copy number changes were observed in the remaining two cases. In one case, in addition to the deletion of 5q14q34 detected by cytogenetic analysis, a loss in copy number on the long arm of 17q at band 17q11.2 encompassing 1.8 Mb including the NF-1 gene was detected by aCGH. Two additional copy number changes in 3q26.1 and 8q24.1 were observed in another case in which monosomy 7 was observed cytogenetically. aCGH failed to detect a clone (2 cells) with a deletion on 5 from band 5p13q13 in one case. In total, 30% of the patients with chromosome analysis displayed an additional cryptic change by aCGH. Our results suggest that a subset of potentially significant genomic alteration may be missed by the conventional chromosome analysis. This pilot study demonstrates that aCGH offers high sensitivity and specificity for routine screening of copy number changes in acute myeloid leukemia.
Novel Multiparametric Quantitative Immunocytomorphological Characterisation of Human Bone Marrow Precursor Differentiation and of Haematopoietic Neoplasms Using ImageStream® Cytometry
Authors: Martin Catherine Claude , Jayat-Vignoles Chantal , Faucher Jean-Luc , George Thaddeus , Venkatachalam Vidya , Morrissey Phil , Feuillard Jean
Published: April 2020

The ImageStream technology performs high speed acquisition of brightfield, laser scatter and up to four fluorescent images per cell for several thousands of cells in suspension, thereby enabling simultaneous immunophenotyping and morphology-based measurements. This is the only technology combining cytology and flow cytometry in one single platform.

Our aim was to study normal and tumour cells of the haematopoietic lineage with this new technology in order to improve diagnosis of haematological disorders. We have defined cytomorphological criteria of normal bone marrow (n=4) and circulating blood cells (n=40). Cells were multi-colour labelled with both DRAQ5 nuclear stain and CD45 ECD-mAb, and additionally labeled with a combination of mAbs against either CD3/CD19, CD11b/CD16, CD14/CRTH2, or CD71/CD235. Results for normal cells were compared to those obtained by classical cytometry and cytology. We then applied these criteria to samples with patients with circulating leukemic cells, including 1 myelodysplatic syndrome (MDS), 1 myeloproliferative syndrome (MPS), 3 acute lymphoblastic leukaemia (ALL), 2 follicular lymphomas (FL) and 20 chronic lymphocytic lymphomas (CLL).

We have created completely new quantitative cytomorphological criteria for classifying blood cells using parameters that measure cellular size and shape, nuclear to cytoplasmic area ratio, nuclear lobe count, SSC texture, the ratio between the size and the major axis of CD45, the ratio between the intensity and the compactness of SSC signal, and the intensity of DRAQ5 labelling, to name a few. Using these criteria, we have characterised normal bone marrow differentiation and normal circulating blood cells. We have obtained a perfect correlation with classical cytology and flow cytometry. Analysis of pathological samples showed that abnormal cells were recognized in all cases. We found an abnormal blast cell compartment and an abnormal monocytic differentiation branch in the case of MDS. We have also defined specific cytomorphological properties that distinguish ALL, FL and CLL tumour cells from normal cells. We also provide data that enumerates the proportion of large cells, of atypical CLL cells and of cells in the G2/M phase.

Altogether, these results show that a technology combining cytology and flow cytometry in a single platform leads to the discovery of completely new and quantitative cytomorphological parameters defining each stage of normal cell and each category of abnormal cells of the haematopoietic lineage, opening completely new perspectives for the diagnosis of haematopoietic neoplasms.
Functional Characterisation of a MYB-HBS1L Intergenic Region Controlling Fetal Hemoglobin Levels in Adults
Authors: Wahlberg Karin E , Jiang Jie , Rooks Helen , Best Steve , Thein Swee Lay
Published: April 2020

A major ameliorating factor in sickle cell disease and beta-thalassemia is the inherent ability to produce fetal hemoglobin (HbF, a2g2), a highly heritable trait. We have previously mapped a major quantitative trait locus (QTL) controlling HbF levels to an array of single nucleotide polymorphisms (SNPs) within a 79 kb block between the gene for HBS1L (a G-protein/elongation factor) and the MYB proto-oncogene in Northern European Caucasians. These variants in chromosome 6q23.3, are referred to as HBS1L-MYB intergenic polymorphism (HMIP) and exist in three linkage disequilibrium (LD) blocks, HMIP-1, -2 and -3, of which HMIP-2 shows the strongest association. Association of HMIP with HbF has recently been replicated in two independent cohorts of patients with sickle cell disease, African-American and Brazilian, as well as in a cohort of Chinese patients with beta-thalassemia. Physiologically, MYB and HBS1L expression was simultaneously down-regulated in individuals with high HbF while over-expression of MYB inhibits gamma-globin expression. We also showed that HMIP influences erythrocyte, platelet and monocyte counts in humans. We hypothesised that the genetic variants in the MYB-HBS1L intergenic region are situated in a regulatory region and affect HbF levels by altering the expression of MYB and/or HBS1L.

Initial DNase I hypersensitivity analysis of the HMIP-2 region in K562 cells identified a number of hypersensitive sites which encouraged further functional analysis of the region. To study activity and to map potential regulatory elements in the entire MYB-HBS1L region we used chromatin immunopreciptation on microarray (ChIP–chip) of acetylated histones H3 and 4, RNA polymerase II and GATA-1. ChIP was performed on chromatin from primary human erythroid precursor cells and enrichment for specific sequences was evaluated using a high resolution Nimblegen microarray covering a large part of chromosome 6q.

Our ChIP-chip data show high levels of histone acetylation in the MYB-HBS1L intergenic region with a concentration in the HMIP-2 block, indicating that the region is transcriptionally active in erythroid precursors. Moreover, a number of potential cis-regulatory elements were identified in the same region as strong GATA-1 signals in coincidence with DNase I hypersensitive sites. These results suggest a regulatory region in the MYB-HBS1L intergenic region that could be important in erythroid development by controlling expression in the MYB locus.