Number Of Red Cells Research Articles

TGF‐b receptor II plays a vital role in neural crest‐derived osteogenesis but not in limb osteogenesis during postnatal development

It is widely accepted that TGF-b signaling enhances osteogenesis via osteoprogenitor enrichment. However, removing TGF-b receptor II (Tgfbr2, a key receptor for TGF-b signaling) in bone cells by osteocalcin-Cre resulted in a controversial long bone phenotype: an increase in trabecular bone but a decrease in cortical bone. To address the role of Tgfbr2 in postnatal mandibular development, we first generated a mouse line in which Tgfbr2 was conditionally deleted in early mesenchymal progenitors by crossing Gli1-CreERT2; Tgfbr2fl/fl; and R26RtdTomato (for cell lineage tracing) compound mice (early cKO). This early cKO line was induced at P5 and harvested at P28. Compared to the control littermates, BV/TV and BMD were significantly reduced in both the cortical bone and alveolar bone of the mandible based on uCT analysis (n = 4; p<0.05). Masson trichrome and polarized light images showed a loss of periodontal ligament fibers and bone mass in the early cKO, whereas there was no apparent change in TRAP positive cells in the cKO bone, excluding the impact of osteoclasts on bone phenotypes. The cell lineage tracing data reveals a great reduction in red bone cell numbers in early cKO. Immunostain images revealed a 70% reduction of OSX in the early cKO bone cells (p < 0.001). Immunostain images also displayed a sharp reduction of bone matrix markers such as MEPE and SOST in the cKO, supporting a requirement of Tgfbr2 in the early osteogenesis. Next, we generated a mouse line in which Tgfbr2 was conditionally deleted in osteoblasts by crossing 3.2 Col1-CreERT2; Tgfbr2fl/fl; R26RtdTomato compound mice (late-cKO). Both uCT and histological data showed mild reduction of bone volume in the late cKO compared to the control littermates. The tracing data revealed little change in bone cell number, and immunostain images showed no apparent alternations in OSX or other bone markers. Importantly, in both cKO mice, there is no apparent change in postnatal long bone development, which suggests that TGF-b signaling plays a more critical role in the neural-crest derived mandible growth. Based on the above data, we conclude that Tgfbr2 in mandible bone formation is mainly required for early bone progenitor cells, and deletion of Tgfbr2 has little impact on the late bone-forming cells during postnatal development of alveolar bone.

Dynamic changes in murine erythropoiesis from birth to adulthood: implications for the study of murine models of anemia.

Liver, spleen, and bone marrow are 3 key erythropoietic tissues in mammals. In the mouse, the liver is the predominant site of erythropoiesis during fetal development, the spleen responds to stress erythropoiesis, and the bone marrow is involved in maintaining homeostatic erythropoiesis in adults. However, the dynamic changes and respective contributions of the erythropoietic activity of these tissues from birth to adulthood are incompletely defined. Using C57BL/6 mice, we systematically examined the age-dependent changes in liver, spleen, and bone marrow erythropoiesis following birth. In addition to bone marrow, the liver and spleen of newborn mice sustain an active erythropoietic activity that is gradually lost during first few weeks of life. While the erythropoietic activity of the liver is lost 1 week after birth, that of the spleen is maintained for 7 weeks until the erythropoietic activity of the bone marrow is sufficient to sustain steady-state adult erythropoiesis. Measurement of the red cell parameters demonstrates that these postnatal dynamic changes are reflected by varying indices of circulating red cells. While the red cell numbers, hemoglobin concentration, and hematocrit progressively increase after birth and reach steady-state levels by week 7, reticulocyte counts decrease during this time period. Mean cell volume and mean cell hemoglobin progressively decrease and reach steady state by week 3. Our findings provide comprehensive insights into developmental changes of murine erythropoiesis postnatally and have significant implications for the appropriate interpretation of findings from the variety of murine models used in the study of normal and disordered erythropoiesis.

Cerebrospinal Fluid (CSF) Analysis of Tumor-Specific Cell-Free DNA (cfDNA) As a Diagnostic and Prognostic Tool for Central Nervous System (CNS) Invasion in Lymphoma

Background: CNS invasion in aggressive lymphoma (including diffuse large B-cell [DLBCL], double-hit [DHL], Burkitt [BL] and plasmablastic lymphoma [PBL]) remains a major diagnostic and therapeutic challenge. Parenchymal CNS invasion requires a brain biopsy to diagnose, and conventional CSF evaluations lack sensitivity. Furthermore, determining which patients (pts) without CNS disease at diagnosis might be at high risk of future CNS relapse, and thus would most benefit from aggressive CNS prophylaxis, is difficult. Predictors that use clinical variables (e.g. the CNS-IPI) can select a high-risk group with ~10% risk of CNS relapse, but ~50% of events occur in low/intermediate-risk groups. We evaluated a novel CSF assay that detects clonotype-specific cell-free DNA (cfDNA) by next-generation sequencing (NGS) combined with multiplex PCR (Chang T et al, BMC Cancer 2020). This assay can identify minimal residual disease (MRD) in the plasma of pts with DLCBL and can predict systemic relapse (Roschewski et al, Lancet Oncol 2015). Methods: To examine the diagnostic sensitivity of the NGS-MRD assay in the CSF, we prospectively collected CSF from 6 pts with lymphomas who had known parenchymal or leptomeningeal CNS invasion. We also tested stored DNA from historical CSF samples of 8 pts with CNS lymphoma who tested negative by conventional IGH-PCR. We then enrolled 19 pts with newly diagnosed high-risk lymphomas without CNS invasion to examine the prognostic value of the NGS-MRD assay in the CSF collected at diagnosis. In the two-step assay, tumor-specific clonotype sequences were first identified from genomic DNA in primary tumor specimens using NGS of rearranged IGK, IGH (VJ or DJ), and IGL loci. Tumor-specific clonotypes were then selected for tracking in the CSF (split into acellular CSF fluid and cell pellet) and in paired plasma samples. The assay quantitated copy numbers of each tracked sequence per mL of acellular CSF or per 106 cells in the pellet, as well as tumor clonotype frequency (%) per all B-cells. Results: The NGS-MRD detected tumor-specific clonotype in 100% of CSF samples from pts with known CNS lymphoma (n=6; Fig A), including pts with isolated parenchymal-only CNS disease that was not detected by CSF cytology, flow cytometry, or IGH-PCR. For pts with CNS parenchymal disease, median cfDNA copy count in the CSF fluid was 2 /mL (range, 0.4-929) and median clonotype frequency was 9.0% (range, 0.03-68.9%). For pts with leptomeningeal disease, these values were 2233 /mL (1.2-5620) and 37% (1.7-98.4%), respectively (Fig. B). Furthermore, the NGS-MRD assay detected tumor clonotypes in DNA isolates from 6 historical CSF samples (collected in 2014-2018) of pts with known CNS disease, in which lymphoma was not detectable by conventional IGH-PCR (2 additional historical samples failed DNA quality check). Among 19 newly diagnosed pts with no known CNS invasion (median age 57, 42% women, median LDH 352 IU/L), 12 (63%) had DLBCL with high risk of CNS relapse by virtue of high CNS-IPI or epidural tumor, and 7 had other high-risk histologies (2 DHL, 2 BL, HIV+ PBL, and 2 lymphoblastic leukemias). The CSF NGS-MRD assay was positive for tumor-specific clonotype in 8 pts (42%). Median cfDNA copy count in the CSF fluid was 3 /mL (range, 0.9-15.6) and median clonotype frequency was 18.6% (range, 1.4-28.5%). We observed no significant correlation between the amount of cfDNA and number of red cells /mL of CSF (a measure of potential CSF contamination by blood during collection procedure; Fig. C) or between the amount of cfDNA in plasma and in CSF (Fig. D). With median follow-up of 11 months, no pts with negative baseline NGS-MRD assay in CSF relapsed, whereas 2 of 10 with a positive assay had a CNS relapse (12-month incidence, 29%; Fig. E), despite both pts having negative CNS imaging, CSF cytology, flow cytometry, and IGH-PCR at diagnosis. Conclusions: In this proof-of-concept study, the NGS-MRD CSF assay showed 100% sensitivity for diagnosing intraparenchymal CNS invasion in aggressive lymphomas that were not detected in the CSF using conventional methods. In a prospective cohort of newly diagnosed pts without baseline CNS invasion but at high clinical risk for CNS relapse, the assay identified lymphoma-derived cfDNA in 42% of cases. The 100% sensitivity and 100% negative predictive value of the assay for subsequent CNS relapse warrants further prospective evaluation as a potential tool for personalizing CNS-directed prophylaxis. Disclosures Olszewski: Spectrum Pharmaceuticals: Research Funding; TG Therapeutics: Research Funding; Adaptive Biotechnologies: Research Funding; Genentech, Inc.: Research Funding. Mullins:Adaptive Biotechnologies: Current Employment, Other: shareholder.

A Novel TBI Free Conditioning Protocol for Haploidentical Transplant in Acquired Aplastic Anemia: (FluCAB-Prime)

Introduction Allogeneic hematopoietic stem cell transplant (HSCT) is the standard treatment for patients younger than 40 years with Severe and Very Severe Aplastic Anemia (AA) who have a Matched Related Donor (MRD). For patients lacking a MRD, treatment options include immunosuppressive therapy (IST), matched unrelated donor (MUD) or alternate donor (haploidentical/cord blood) transplant. Pakistan has a population of around 23 million but there is no donor registry for MUD transplants and horse antithyomcyte globulin (hATG) is not available. Over past decade, results of haploidentical transplants have improved remarkably with use of post-transplant cyclophosphamide. However, most of these protocols incorporate total body irradiation (TBI) to improve engraftment and reduce graft failure. TBI is not available in most of the transplant centres across Pakistan. We have developed a novel TBI free conditioning regimen (NCT03955601) for haploidentical HSCT in acquired AA patients lacking a MRD. Materials and Methods We conducted a prospective, single centre, interventional trial (NCT03955601) using novel TBI free conditioning at AF bone marrow transplant centre (AFBMTC)/ National institute of blood and marrow transplant (NIBMT) for patients with acquired severe and very severe AA. Between July 2018 and March 2020, a total of 10 patients received related haploidentical transplant.Study inclusion criteria was diagnosis of severe and very severe AA, patients of both genders, age 2-60 years, Karnofsky performance status&amp;gt;70%. Exclusion criteria was presence of donor specific antibodies (DSA), inherited bone marrow failure syndrome, prior HSCT and severe sepsis. Conditioning regimen used was Fludarabine (Flu) 30 mg/m2 IV daily from day -7 to -3, Cyclophosphamide (Cy) 14.5 mg/kg IV daily on day -6 and -5 , rabbit Antithymocyte globulin (rATG) 5 mg /kg/day from day -6 to day-3; Busulphan (Bu) IV 3.2 mg per kg/day in 04 divided doses on day -3 and day-2, Granulocyte Colony Stimulating factor (GCSF) primed Bone marrow harvest (BMH) and/or PBSC graft on day 0 and day +1 respectively. Graft versus host disease (GVHD) prophylaxis used was post-transplant cyclophosphamide (PTCy) administered at a dose of 50mg/kg/day given daily on days +3 and +5 post-transplant, cyclosporine (CsA) from day +5 and mycophenolate mofetil (MMF) from day+5 to +35. Primary outcome measure was overall survival (OS) while secondary outcome measures included graft failure, treatment related mortality (TRM), disease free survival (DFS), GVHD free relapse free survival (GRFS), acute and chronic GVHD. Results: Ten patients were transplanted, 5 (50%) female and 5 (50%) male (table 1). Median age was 15.5 years (range 5-41 years). Median duration from diagnosis to transplant was 14 months (range 4-51 months). One patient received ATG prior to transplant. Median number of red cell concentrate (RCC) transfusions before transplant were 27 (8-90) and platelet transfusions 100(6-150). Median donor age was 23 years (10-41 years). Donor-recipient major ABO mismatch was present in 2(20%) while four (40%) had minor ABO mismatch. Primed bone marrow harvest (BMH) was used in 3(30%) while primed BM+PBSC was given in 7(70%) patients. Median CD34 dose given was 8 x 106/kg (range 5.1-16). Seven patients (70%) achieved sustained neutrophil engraftment. One patient had primary graft failure, one patient secondary graft failure at day 35 due to Cytomegalovirus infection and one patient (currently day +118) is having poor graft function. Acute skin GVHD stage-2 developed in 1 patient which settled with topical steroids. None of the patient developed chronic GVHD. Cytomegalovirus reactivation was documented in 8 (80%) patients. All donors and recipients were CMV seropositive pre-transplant. One patient (female, 20 years) had primary graft failure and died on day +31 with sepsis and multiorgan dysfunction syndrome (MODS). One patient (female, 14 years) had secondary graft failure due to CMV infection and died on day +44 with intracranial hemorrhage. Conditioning regimen was well tolerated without any treatment related morbidly and mortality. Median follow-up of study was 13 months (range 4-22 months). Overall survival of study cohort is 80%, DFS 70% and GRFS 70%. Conclusion: Our study shows that for countries lacking TBI, use of FluCAB-Prime protocol is feasible and is associated with low rates of acute and chronic GVHD. Disclosures No relevant conflicts of interest to declare.

Key Determinants of Anemia among Youngsters under Five Years in Senegal, Malawi, and Angola.

Anemia is characterized as a condition where there is a deficient number of hematocrit, hemoglobin, or red cells in the human body. This condition affects most youngsters under five years old and pregnant women. The fundamental goal of this paper is to investigate anemia, recognize its determinants, and propose critical proposals to achieve 2030 Sustainable Development Goal with a focus on Senegal, Malawi, and Angola. This research utilized 2016 nationally representative information from Senegal, Malawi, and Angola, which involved collecting data on the demographic and health of the populaces. The Demographic and Health Survey information from Senegal, Malawi, and Angola was then merged to create a pooled sample. This statistical technique enables to generalize and compare the results. A generalized linear mixed model was utilized to decide the factors correlated with anemia among youngsters under five years in Senegal, Malawi, and Angola. The analysis was performed in SPSS and SAS software. A generalized linear mixed model results showed that, compared to youngsters aged less than 12 months, youngsters in the age interval 13–23, 24–35, 36–47, and 48–59 months are more likely to be affected by anemia (OR = 1.419, 2.282, 3.174 and 4.874 respectively). In this study, seven factors were included in the final model. However, only five were found to be significant in explaining anemia at the 5% level of significance. The generalized linear mixed model identified youngster’s age, gender, mother’s level of schooling, wealth status, and nutritional status as determinants of anemia among youngsters under five years in Senegal, Malawi, and Angola.

Near Infrared Spectroscopy in Anemia Detection and Management: A Systematic Review.

Red cell transfusions are intended to improve oxygen delivery to tissues. Although studies comparing hemoglobin concentration triggers for transfusion have been done, the hemoglobin threshold for clinical benefit remains uncertain. Direct measurement of tissue oxygenation with non-invasive near infrared spectroscopy has been proposed as a more physiological transfusion trigger, but its clinical role remains unclear. This systematic review examined the role of near infrared spectroscopy for detection of anemia and guiding transfusion decisions. Abstracts were identified up until May 2019 through searches of PubMed, EMBASE and The Web of Science. There were 69 studies meeting the inclusion criteria, most (n = 65) of which were observational studies. Tissue oxygen saturation had been measured in a wide range of clinical settings, with neonatal intensive care (n = 26) and trauma (n = 7) being most common. Correlations with hemoglobin concentration and tissue oxygenation were noted and there were correlations between changes in red cell mass and changes in tissue oxygenation through blood loss or transfusion. The value of tissue oxygenation for predicting transfusion was determined in only four studies, all using muscle oxygen saturation in the adult trauma setting. The overall sensitivity was low at 34% (27%-42%) and while it had better specificity at 78% (74%-82%), differing and retrospective approaches create a high level of uncertainty with respect to these conclusions. There were four prospective randomized studies involving 540 patients, in cardiac and neurological surgery and in neonates that compared near infrared spectroscopy to guide transfusion decisions with standard practice. These showed a reduction in the number of red cells transfused per patient (OR: 0.44 [0.09-0.79]), but not the number of patients who received transfusion (OR: 0.71 [0.46-1.10]), and no change in clinical outcomes. Measuring tissue oxygen saturation has potential to help guide transfusion; however, there is a lack of data upon which to recommend widespread implementation into clinical practice. Standardization of measurements is required and greater research into levels at which tissue oxygenation may lead to adverse clinical outcomes would help in the design of future clinical trials.

1001 – REGULATION OF HEMATOPOIETIC PROGENITOR CELL DIVISION: DEVELOPMENT OF THERAPIES FOR ERYTHROPOIETIN- RESISTANT ANEMIAS

Many anemias are not treatable with Epo because CFU-E progenitors are too few in number to maintain sufficient red blood cell production. Treatment requires a drug that acts at an earlier stage to enhance formation of CFU-Es. We showed that glucocorticoid receptor (GR) agonists, used to treat such anemias despite side effects, increase erythroid output by expanding the number of BFU-E progenitor cells; glucocorticoids stimulate additional BFU-E divisions leading over time to an increase in numbers of CFU-Es and mature red cells. We showed that activation of PPARα by agonists used for dyslipidemia synergizes with the GR to promote BFU-E cell expansion and increase erythrocyte production. In BFU-Es PPARα co-occupies many chromatin sites with the GR and stimulates transcription of an overlapping set of genes essential for self-renewal divisions. We suggest that clinically tested PPARα agonists may improve the treatment of Epo resistant anemias. Inhibitors of TGF-b signaling also induce extra BFU-E divisions and could also be used. BFU-E cells are heterogenous with respect to the sizes of colonies they form. We used single cell RNA sequencing to demonstrate a continuum of transcriptomic states in BFU-Es, concordant with a continuum of their proliferative potentials. Glucocorticoids do not trigger BFU-E self-renewal but reduce the rate of progression along this developmental continuum without changing the rate of cell division. Glucocorticoids drive prolonged expression of genes known to antagonize terminal erythroid differentiation and slow induction of genes that drive terminal differentiation. In both the presence and absence of glucocorticoids, progenitor cell division is largely symmetric as both daughter cells have similar transcriptomes. Thus the rate of progression along the erythroid developmental continuum dictates the absolute number of mature erythrocytes generated from each early BFU-E progenitor. These findings suggest a paradigm for control of the magnitude of cellular output in many other developmental pathways with transcriptional continuums.

3085 – THE EPOR STIMULATES RAPID CYCLING AND FORMATION OF LARGER RED CELLS DURING MOUSE AND HUMAN ERYTHROPOIESIS

EpoR signaling is essential for the survival of erythroid precursors during terminal differentiation, when these cells undergo 3-5 cell divisions that are coupled to a rapid loss in cell size. The absolute dependence of erythroblasts on EpoR signaling for survival makes it challenging to identify other essential functions of EpoR in these cells. Key open questions include a role for EpoR in cell cycle regulation; and a potential role for EpoR in the induction of erythroid genes. To address these gaps, we developed a genetic system that allowed us to examine essential non-survival functions of EpoR signaling in erythroid differentiation. We rescued mouse Epor-/- fetal liver erythroid progenitors from apoptosis by transduction with the anti-apoptotic protein bcl-xL, comparing their ensuing differentiation with that of Epor-/- progenitors that were rescued by re-introduction of the EpoR. We found that the bcl-xL survival signal, in the absence of any EpoR signaling, was sufficient to allow erythroid terminal differentiation and formation of anucleated red cells. However, key features were abnormal. First, erythroblasts underwent slower and fewer cell cycles, generating fewer than half the number of red cells, showing an essential role for EpoR signaling in the regulation of cell cycle speed and number. Second, we found that EpoR signaling unexpectedly increased red cell size, in spite of the concurrent increase in the number of cell divisions. Using mice doubly deleted for both EpoR and HRI, we found that EpoR regulation of red cell size is independent of the well-described red cell size regulation by iron and heme. Healthy human volunteers that were administered Epo showed an increase in mean corpuscular volume (MCV) that persisted long after Epo and reticulocyte levels returned to baseline, supporting a role for the regulation of red cell size by Epo and suggesting new diagnostic interpretations for this common clinical test. Together, our findings reveal novel functions for the EpoR in erythroid differentiation including an unexpected role in the regulation of red cell size.

P0227VITAMIN K ANTAGONIST-ASSOCIATED MICROSCOPIC HEMATURIA: ASSOCIATION WITH INR LEVELS AND PROTECTIVE EFFECT OF RAAS BLOCKADE

Abstract Background and Aims Vitamin K antagonists (VKA) are the most widely used anticoagulants in the daily clinical practice for the prevention of thrombotic events. Several renal adverse effects have been associated with the use of VKA, from hemodynamic effects due to massive urological hemorrhage associated with VKA overdose, to non-hemodynamic effects that include glomerular hemorrhage and renal tubular obstruction due to red cell casts. The main aim of our study was to establish an association between INR levels and microscopic hematuria in patients with VKA, and identify possible risk and protective factors associated with development of microscopic hematuria in these patients. Method We performed a transversal study that included patients with oral anticoagulation with coumarin derivatives (warfarin or acenocoumarol), that attended the Hematology outpatient clinic from October to December 2019 for routine INR control. A urine specimen was collected on the day of the INR control. We performed a simple urinalysis to all patients and quantified the precise number of red cells in the urine sediment by flow cytometry. Demographic data, kidney function tests including serum creatinine and albuminuria, comorbidities, anticoagulant dose and concomitant treatment were registered. Results 337 patients were included in the study, with an average age of 68.6 ± 12.2 years, 51% were females, 95% were treated with acenocoumarol and 5% with warfarin. Median INR levels were 2.6 (IQR 2.1-3.3) and the median number of urine red cells in the sediment were 11 RBCs/µl (IQR 0-13). 11.9% of the patients presented microscopic hematuria (≥14 RBCs/µl). There was a significant correlation between INR levels and the number of RBCs in the urine sediment (Spearman’s rho= 0.119, p= 0.029), independent from the type of anticoagulant used. This correlation was stronger after excluding those patients who had concomitant bacteriuria (Spearman’s rho= 0.201, p= 0.024). We found no correlation between time elapsed from start of anticoagulation or total weekly dose with the intensity of microscopic hematuria. In the univariate analysis, microhematuria was associated with having an INR &amp;gt;3.5 (19% vs. 10.2%, p=0.046), bacteriuria (15.2% vs. 3.6%, p=0.015), leukocyturia (14.8% vs. 6.6%, p= 0.026), hypertension (16.2% vs. 9.5%, p=0.053), and the use of RAAS blockers (17.2% vs. 6.9%, p=0.004). After performing a multivariate logistic regression that included the aforementioned variables, the association between microhematuria and RAAS blockade with either an ACE inhibitor or ARB was maintained (OR 0.38, CI 95% 0.163-0.886, p=0.025), independent from INR levels, hypertension, leukocyturia or bacteriuria. We found no association between serum creatinine, albuminuria or antiplatelet treatment with the presence of microscopic hematuria. Conclusion INR overdose is significantly associated with the presence of microscopic hematuria, independent from the type of VKA used or the total weekly dose. RAAS blockade is an independent protective factor for the presence of microscopic hematuria in anticoagulated patients.

Hematological Impacts of the Traffic Emissions in Sulaymaniyah Governorate

Abstract&#x0D; The current study was achieved to evaluate the essential hematologic impacts of traffic emission. Ninety-six cases were studied included both exposures and controls. The focal point was on Raparin District in Sulaimani Governorates. A questioner form was depended for collecting information about each case. Fresh venous blood (5ml) were collected aseptically from both exposures and controls. Hematologic Autoanalyzer (Coulter) was used for hematologic investigations. It appeared that the mean leukocyte counts were higher among exposures in comparison to controls; the period of exposure and smoking were significantly effective on total white cells. Lymphocyte counts were significantly declined among exposures. It appeared that the distance from the emission gas sources, smoking, and period of exposure was significantly effective on the total lymphocyte counts (p&lt;0.05). No valuable effects of traffic emission were noticed on granulocytes in general (p&gt;0.05), although the neutrophil counts were significantly higher among exposure. It was concluded that traffic emission has no significant effects on Red cell profiles, including red cell numbers, Hb, HCT, RDW, MCH in general (p&gt;0.05). The current study revealed that there were no noticeable effects of traffic emission, generally on the total platelet counts between exposures and controls. It has concluded that the distance from the emission sources was significantly effective on platelet counts among exposures themselves (p&lt;0.05).

Clinical application of preoperative selective arterial embolization for spinal tumors

Objective To investigate the clinical value of preoperative selective arterial embolization for spinal tumors. Methods The clinical data of 42 consecutive patients who underwent spinal tumor resection in department of orthopedics Shengjing Hospital of China Medical University from January 2017 to December 2018 were retrospectively analyzed. Patients were divided into embolization group (20 cases) and non-embolization group (22 cases) according to whether they underwent arterial embolization before tumor resection. Two surgical treatments including vertebral resection and laminectomy were performed. The embolization group included 12 cases of vertebral resection and 8 cases of laminectomy; while the non-embolization group included 13 cases of vertebral resection and 9 cases of laminectomy. The difference of intraoperative estimated blood loss, total number of transfused packed red blood cell, calibrated estimated blood loss, operation time and hospitalization time were compared using independent sample t test. Results Twenty patients in the embolization group underwent successful interventional embolization without serious complications. There were no significant differences between the embolization group and the non-embolization group in terms of intraoperative estimated blood loss, total number of transfused packed red blood cell, calibrated estimated blood loss, operation time, and hospitalization time (P>0.05). Among the patients who underwent vertebral resection, intraoperative estimated blood loss, total number of transfused packed red blood cell and calibrated estimated blood loss were (1 966.7±898.8) ml, (7.42±3.27) U and (91.3±39.2) g/L in the embolization group, and (2 838.5±1 143.5) ml, (11.04±4.08) U and (133.0±46.4) g/L in the non-embolization group, respectively, with statistically significant differences (t=-2.107, -2.436, -2.419, P 0.05). Among the patients who underwent laminectomy, there were no significant differences in the above-mentioned indicators between the embolization group and the non-embolization group (P>0.05). Conclusion Selective arterial embolization for spinal tumors is a relatively safe interventional procedure. Preoperative embolization does not significantly reduce the amount of blood loss during surgical procedures. After the surgical procedures were differentiated, preoperative embolization significantly reduces the amount of blood loss in patients underwent vertebral resection, while patients who underwent laminectomy do not benefit significantly. Key words: Neoplasms; Spine; Embolization, therapeutic; Preoperative period; Blood loss, surgical

Successful Personalization of Propylene Glycol Free Melphalan (PGF-MEL) for Multiple Myeloma (MM) and AL Amyloidosis (AL) Patients Undergoing Autologous Hematopoietic Stem Cell Transplant (AHCT) Using Pharmacokinetic (PK)-Directed Dosing

Background Body surface area-based dosing of melphalan prior to AHCT for MM and AL patients causes inter-patient variability in exposure measured by area under the curve (AUC). Higher AUC is associated with increased toxicity, but prolonged survival (Shaw et al., BBMT 2012). Using Evomela (PGF-MEL, Acrotech Biopharma LLC), a more stable and possibly less toxic intravenous formulation of melphalan, we examined the feasibility of PK-directed dosing to individualize and optimize therapy. Methods Prospectively, patients received 100mg/m2 of PGF-MEL on day -2 prior to AHCT. Serum samples were collected at 5, 15, 30, 40, 75, and 150 min after the dose and AUC PK modeling (Phoenix WinNonlin v6.4) was used to determine the dose required to achieve a target AUC of 13.5mg/L*h (+/-1). On day -1, patients received the balance of the calculated dose with samples collected at the same timepoints to calculate the total AUC. After 5 patients were enrolled, the protocol was amended to increase the maximum allowable dose to the equivalent of 260mg/m2 from 220mg/m2. On day +1, 6, 11, 30, 60, and 90, patients reported symptom burden was collected using the MD Anderson Symptom Inventory – MM (MDASI-MM). Results Results of 15 patients are presented with 11 having MM and 4 AL, 60% male, median age 62yr (range 48-71), median body surface area 1.9 (range 1.5-2.2), and median creatinine on day of Mel 0.8mg/dL (range 0.7-1.6). Target doses were calculated and given in 100% of patients. Thirteen of 15 patients (87%) achieved the target AUC. The median AUCs on day -2 and day -1 were 6.3 mg/L*h (range 4.2-11.4) and 5.6 (range 3.1 – 8.4) respectively, with a median total AUC of 13.2 (range 9.6-15.2, Figure 1). Three patients received the capped dose of 220mg/m2 prior to the amendment and achieved their predicted AUC; 1 patient received the minimum 140mg/m2 and also achieved the predicted AUC which was higher than the target AUC, as expected. 9/15 patients had AUCs between 12.5-14.5 with 4/6 patients out of range for the above reasons. Neutropenic fevers occurred in 40% and grade 2 diarrhea for at least 1 day in 80% (median 2 days, range 0-7). Median time to neutrophil engraftment was 9 days (range 9-12). The median number of red cell and platelet transfusions required were 0 (range 0-3) and 1 (range 0-8), respectively. Engraftment syndrome was seen in 30% of patients. The hematologic response rate post AHCT was 100%, including complete remission in 20% and ≥ very good partial response 73%. With median follow-up of 6.4 months (range 2.8-13.1 months), one patient with MM whose AUC was below the target (10.1 units) has relapsed. No patient has died. Conclusion We show for the first time that personalization of therapy to a target AUC using PK-directed PGF-MEL was logistically feasible and prevented underdosing. Full trial results for 35 patients, as well as patient-reported outcomes and toxicities will be presented at the meeting.

Impact of 2,4-D and potato (Solanum tuberosum L.) tuber age on anthocyanin content of skin and phellem anatomy of Red Norland

Attractive appearance is highly desirable for fresh market red-skinned potatoes. Red potatoes derive their skin color from anthocyanins. Some growers attempt to enhance red potato skin color by treating vines with the synthetic auxin 2,4-D. In this study, 2,4-D was applied twice at early bloom stage to ‘Red Norland’ potatoes and tubers were harvested weekly until plants senesced. 2,4-D rapidly increased anthocyanin quantity and altered anthocyanin composition. Average tuber mass at the final harvest was less for the 2,4-D-treatment than untreated controls. Elevated anthocyanin content resulting from 2,4-D persisted through 3 months storage. 2,4-D-treated potatoes did not differ from untreated controls in the number and depth of red pigmented cells in the phellem. Thus, changes in periderm cell division and expansion may not contribute to the 2,4-D response of ‘Red Norland’. Instead, the data suggest that 2,4-D application initiated a rapid change in anthocyanin accumulation that persisted into storage.

Racetrack exercise vs treadmill exercise with respect to exercise-induced pulmonary haemorrhage (EIPH): implications for studies of putative treatments of EIPH

Multiple treadmill-based studies using low numbers of horses have evaluated potential prophylactic treatments for exercise-induced pulmonary haemorrhage (EIPH) and found no effect. However, the relevance of these findings to racing is unclear. Because severity of EIPH incurred on treadmills has not been compared to that following high-speed racetrack exercise in the same horses, we retrospectively performed this comparison using bronchoalveolar lavage fluid red cell numbers (BALFRBC) due to the relative insensitivity of tracheobronchoscopy. Six race-fit Thoroughbreds with recent tracheobronchoscopic EIPH scores ≥2 were exercised to fatigue on a treadmill at 115% V̇O2max (5% incline, 12.3-14.2 m/s), and maximally on a racetrack over 800 m and 1,100 m with average speeds ranging from 16.4-16.7 and 15.5-16.6 m/s, respectively. Run order varied but was not randomised. Bronchoalveolar lavage (BAL) was performed blindly using Bivona tubes 45-60 mins post-exercise. BALFRBC were determined using a haemocytometer. Data were expressed as median and interquartile range, and analysed using RM ANOVA with significance set at P&lt;0.05. BALFRBC were greater after both racetrack runs than after treadmill exercise (P&lt;0.05; treadmill: 10,305/μl (3,871-26,079); 800m: 25,000/μl (17,175-73,400); 1,100m: 19,500/μl (8,962-800,600). Treadmill exercise resulted in lower numbers and a narrower range in BALFRBC than racetrack exercise. Thus, when a small number of horses is used to study EIPH treatments on a treadmill, a lower BALFRBC would be anticipated following the baseline run than with a similar study using racetrack exercise, and might reduce the likelihood of demonstrating significant treatment effects. Results of this retrospective study raise concern regarding the advisability of extrapolating conclusions regarding efficacy of EIPH treatments from treadmill studies to racetrack scenarios.

Allo-Immunisation in Sickle Cell Patients in a Large British Specialist Haemoglobinopathy Centre

Background North Middlesex University Hospital (NMUH) is a 400 bed acute hospital in London, United Kingdom. The hospital is a specialist centre for the treatment of inherited red cell disorders caring for approximately 500 adult patients with Sickle Cell Anaemia (SCA) and performing a large number of emergency and elective blood transfusions including automated exchange transfusions, which are an increasingly important cornerstone in the treatment of SCA. In order to minimise risks of allo-immunisation, international guidelines recommend that sickle cell patients are fully phenotyped prior to first transfusion and receive red cell units that are ABO, Rh (DCcEe) and K matched. This has been implemented at NMUH since the 1990s. However, there is concern that the rate of generation of allo-antibodies is still significant. Methods NMUH undertook a retrospective cross-sectional survey of all currently registered adult (&gt;18 years old) patients with haemoglobin genotypes SS and SC, using local transfusion records dating back to 1991. The Laboratory Information Management System (LIMS) was interrogated to provide the current antibody status of each patient as well as the number of red cells received. To account for transfusions before 1991, outpatient annual review data was examined where numbers of units transfused were recorded. Multivariate logistic regression models were used to explore the relationship between the presence of allo-antibodies and the following variables: sex, age, genotype, cumulative number of transfusions and pregnancy. Results A total of 336 patients with HbSS (n=207) and HbSC (n=129) were identified. The mean age was 39. Male to female split was 43% to 57%. In the HbSS group, 73% (n=173) had received at least one blood transfusion. This was 40% (n=52) in the HbSC group. Number of units received ranged from 1 to greater than 500 for those on regular exchange programmes. An estimate of the number of cumulative red cell units given is shown in Figure 1. 18% (n=42) of transfused patients had alloantibodies ranging from 1 (50% of patients) to a maximum of 5 antibodies). 79% (n=33) of those with allo-antibodies were female. 76 antibodies were identified in total and the breakdown of antibody frequency is shown in Figure 2. 38% (n=29) of antibodies were directed against antigens of the MNSs/Fy system and 33% (n=25) were Rh and /K directed. Multivariable regression showed female sex (OR = 3.54; CI 1.55 - 9.00), age (OR = 1.03; CI 1.00 - 1.05) and number of units transfused (OR = 1.40; CI 1.18 - 1.65) were all independent predictors for antibody formation (p&lt;0.05). Subgroup regression analysis of the female patients showed no impact of pregnancy (p&gt;0.05). Conclusion We presented retrospective data from a large cohort of patients with SCA. Limitations included the fact that transfusions prior to 1991 could not be retrieved from the LIMS and were recorded based on annual review data, which although comprehensive, cannot guarantee to fully account for transfusions received elsewhere. The data suggests that despite transfusing in accordance with international guidelines, the level of allo-immunisation remains significant and is particularly marked in females. The majority consisted of potentially hazardous antibodies (Rh/K/Ss/Fy), with only 18% being of limited clinical importance (M/Le). The relatively high frequency of anti-Rh/K requires further investigation to ascertain if these are due to phenotype/genotype mismatch or due to transfusion of inappropriate units at other centres. The high incidence of Ss/Fy antibodies is in line with findings from other papers [Compernolle et al, Transfusion 2018;58;1555-1566] and warrants further research into selecting Ss/Fy compatible units, especially in the younger generation of SCA patients. Disclosures No relevant conflicts of interest to declare.

Effects of Krüppel-like factor 6 overexpression towards apoptosis of human lens epithelial cells with ultra violetradiation B treatment

Objective To investigate the effect of the overexpression of Kruppel-like factor 6 (KLF6) towards the apoptosis of human lens epithelial cells (HLECs) induced by ultraviolet B (UVB) radiation. Methods The eukaryotic expression plasmid pEGFP-C2-KLF6 which was successfully constructed were transfected into HLECs, followed by the detection of KLF6 level by using Western blot, and then companied by UVB stimulation.Cell viability was measured by methyl thiazolyl tetrazolium (MTT) assay.The morphology of the cells was observed by using hematoxylin-eosin staining method.The cell damage was examined by Live/Dead staining.The apoptotic markers bax and bcl-2 were detected by Western blot.Quantitative apoptotic levels were measured with the apoptosis detection kit; the expression level of reactive oxygen species (ROS) was analyzed by DCFH-DA probe. Results The cell viability of the 0.5 μg transfection group and the 1.0 μg transfection group was significantly lower than that of the blank vector control group (both at P<0.05). In high KLF6 expression group, the cells were sparse, long and narrow in size and shape, and the cytoplasm was concentrated.The cells in the normal control group were green living cells with stable morphology and even quantity.The number of red dead cells was increased significantly in the KLF6 high-expression group.After UVB irradiation, the apoptosis value, relative bax expression, bax/bcl-2 ratio and ROS expression of HLECs cells in the KLF6 high-expression group were all higher than those in the blank vector control group, with statistically significant differences between them (all at P<0.05). Conclusions Overexpression of KLF6 can exacerbate apoptosis of HLECs caused by UVB, by regulating the expression of apoptosis-related proteins and promoting the accumulation of ROS in the endoplasmic reticulum.Down-regulation of KLF6 expression by biological tools may play a protective role on LECs to a certain extent. Key words: Kruppel factor 6; Lens epithelial cells; Apoptosis; Ultra violetradiation B; Oxidative stress

Correlation of Thromboelastography Parameters with Bleeding Episodes of Post-LVAD Surgery

Purpose Left ventricular assist device (LVAD) is considered as an alternative treatment for patients with refractory end-stage heart failure. Perioperative bleeding is one of the most common adverse events of LVAD surgery. Thromboelastography (TEG; Haemoscope, Niles, IL, USA), a commercial assay that assesses clot formation, has been shown to reduce blood product use in cardiac surgeries. However, the information in LVAD surgery is very minimal. We aim to assess the statistical correlation between TEG parameters and bleeding during perioperative period. Methods We conducted a retrospective study of patients who underwent LVAD implantation at University Medical Center, Lubbock, Texas from July 1st, 2016 to July 31st, 2018. We excluded the patients whose TEG was not checked prior to LVAD surgery. We collected maximal amplitude of clot formation in response to arachidonic acid (MAAA), clot strength (GAA) and bleeding outcomes. Results Sixteen LVAD patients were included in the study with mean age of 57.2±8.2 years old (see table 1 for baseline characteristics and lab values). MAAA and GAA showed significant negative correlation with surgical drain output, number of packed red cell units and number of platelet units (p = 0.04, 0.04 and 0.01 respectively for MAAA and p = 0.04, 0.03 and 0.01 respectively for GAA). MAAA and GAA also showed moderate negative correlation with length of hospital stay and episodes of surgical re-exploration due to bleeding (r = -0.47 and -0.42 for MAAA and r = -0.43 and -0.42 for GAA) although without statistical significance (p ≥ 0.05). Conclusion Our study found that TEG parameters correlated significantly with drain output, number of packed red cell and platelet units used during perioperative period. They also tended to correlate with the hospital length of stay and surgical re-exploration but were likely limited by small number of study population. Further studies with larger population are needed for validation of the results.

Blood image analysis to detect malaria using filtering image edges and classification

Malaria is a most dangerous mosquito borne disease and its infection spread through the infected mosquito. It especially affects the pregnant females and Children less than 5 years age. Malarial species commonly occur in five different shapes, Therefore, to avoid this crucial disease the contemporary researchers have proposed image analysis based solutions to mitigate this death causing disease. In this work, we propose diagnosis algorithm for malaria which is implemented for testing and evaluation in Matlab. We use Filtering and classification along with median filter and SVM classifier. Our proposed method identifies the infected cells from rest of blood images. The Median filtering smoothing technique is used to remove the noise. The feature vectors have been proposed to find out the abnormalities in blood cells. Feature vectors include (Form factor, measurement of roundness, shape, count total number of red cells and parasites). Primary aim of this research is to diagnose malaria by finding out infected cells. However, many techniques and algorithm have been implemented in this field using image processing but accuracy is not up to the point. Our proposed algorithm got more efficient results along with high accuracy as compared to NCC and Fuzzy classifier used by the researchers recently.

Time course of peripheral blood count recovery during induction chemotherapy for childhood acute lymphoblastic leukemia

ABSTRACTIntroduction: Children with newly diagnosed acute lymphoblastic leukemia (ALL) present with low peripheral blood counts caused by bone marrow replacement. The recovery of counts during induction chemotherapy is not well described.Material and methods: Records for 63 children with ALL were reviewed. Peripheral hematology blood counts during five weeks of induction chemotherapy were extracted, and the time to partial recovery with safe counts and complete recovery with normal counts in the three cell lines determined. The number of red cell and platelet transfusions, the number of febrile episodes, and the number of days on intravenous antibiotics were counted.Results: Platelet recovery occurred early: median time to achieving counts >50/nL 14 days, to counts >100/nL 16 days. Neutrophil recovery was relatively slow: median time to counts >0.5/nL 18 days, to counts >1.0/nL 26 days. The time to partial recovery was shorter in high risk than in lower-risk treatment groups. Partial platelet recovery by day 15 indicated early recovery and lower morbidity. Complete platelet recovery day 15 was significantly associated with residual disease <0.1% after four weeks. Lymphocyte counts showed a marked decrease in first two weeks followed by a rise in the next three weeks; a count <0.35/nL on day 15 was associated with poor response.Conclusion: After starting chemotherapy for ALL, platelet recovery can be expected after two to three weeks while neutrophil recovery lasts three to five weeks. Platelet and lymphocyte counts after two weeks treatment may give an indication of residual disease after four weeks.

Hemoglobin Changes After Long-Term Intermittent Work at High Altitude

Chronic high altitude hypoxia leads to an increase in red cell numbers and hemoglobin concentration. However, the effects of long-term intermittent hypoxia on hemoglobin concentration have not fully been studied. The aim of this study was to evaluate hemoglobin levels in workers commuting between an elevation of 3,800 m (2-week working shift) and lowland below 1,700 m (2 weeks of holiday). A total of 266 healthy males, aged from 20 to 69 years (mean age 45.9 ± 0.6 years), were included into this study. The duration of intermittent high altitude exposure ranged from 0 to 21 years. Any cardiac or pulmonary disorder was excluded during annual check-ups including clinical examination, clinical lab work (blood cell count, urine analysis, and biochemistry), ECG, echocardiography, and pulmonary function tests. The mean hemoglobin level in workers was 16.2 ± 0.11 g/dL. Univariate linear regression revealed an association of the hemoglobin levels with the years of exposure. Hemoglobin levels increased 0.068 g/dL [95% CI: 0.037 to 0.099, p < 0.001] for every year of intermittent high altitude exposure. Further, after adjusting for other confounding variables (age, living at low or moderate altitude, body mass index, and occupation) using multivariable regression analysis, the magnitude of hemoglobin level changes decreased, but remained statistically significant: 0.046 g/dL [95% CI: 0.005 to 0.086, p < 0.05]. Besides that, a weak linear relationship between hemoglobin levels and body mass index was revealed, which was independent of the years of exposure to high altitude (0.065 g/dL [95% CI: 0.006 to 0.124, p < 0.05]). We concluded that hemoglobin levels have a linear relationship with the exposure years spent in intermittent hypoxia and body mass index.