Perl's Stain Research Articles

Colocalization of Erythrocytes and Vascular Calcification in Human Atherosclerosis: A Systematic Histomorphometric Analysis.

Background Intimal calcification typically develops in advanced atherosclerosis, and microcalcification may promote plaque progression and instability. Conversely, intraplaque hemorrhage and erythrocyte extravasation may stimulate osteoblastic differentiation and intralesional calcium phosphate deposition. The presence of erythrocytes and their main cellular components (membranes, hemoglobin, and iron) and colocalization with calcification has never been systematically studied. Methods and Results We examined three types of diseased vascular tissue specimens, namely, degenerative aortic valve stenosis ( n = 46), atherosclerotic carotid artery plaques ( n = 9), and abdominal aortic aneurysms ( n = 14). Biomaterial was obtained from symptomatic patients undergoing elective aortic valve replacement, carotid artery endatherectomy, or aortic aneurysm repair, respectively. Serial sections were stained using Masson–Goldner trichrome, Alizarin red S, and Perl's iron stain to visualize erythrocytes, extracelluar matrix and osteoid, calcium phosphate deposition, or the presence of iron and hemosiderin, respectively. Immunohistochemistry was employed to detect erythrocyte membranes (CD235a), hemoglobin or the hemoglobin scavenger receptor (CD163), endothelial cells (CD31), myofibroblasts (SMA), mesenchymal cells (osteopontin), or osteoblasts (periostin). Our analyses revealed a varying degree of intraplaque hemorrhage and that the majority of extravasated erythrocytes were lysed. Osteoid and calcifications also were frequently present, and erythrocyte membranes were significantly more prevalent in areas with calcification. Areas with extravasated erythrocytes frequently contained CD163-positive cells, although calcification also occurred in areas without CD163 immunosignals. Conclusion Our findings underline the presence of extravasated erythrocytes and their membranes in different types of vascular lesions, and their association with areas of calcification suggests an active role of erythrocytes in vascular disease processes.

Cerebral Microbleeds in Fragile X-Associated Tremor/Ataxia Syndrome.

Fragile X-associated tremor/ataxia syndrome is a neurodegenerative disease of late onset developed by carriers of the premutation in the fragile x mental retardation 1 (FMR1) gene. Pathological features of neurodegeneration in fragile X-associated tremor/ataxia syndrome include toxic levels of FMR1 mRNA, ubiquitin-positive intranuclear inclusions, white matter disease, iron accumulation, and a proinflammatory state. The objective of this study was to analyze the presence of cerebral microbleeds in the brains of patients with fragile X-associated tremor/ataxia syndrome and investigate plausible causes for cerebral microbleeds in fragile X-associated tremor/ataxia syndrome. We collected cerebral and cerebellar tissue from 15 fragile X-associated tremor/ataxia syndrome cases and 15 control cases carrying FMR1 normal alleles. We performed hematoxylin and eosin, Perls and Congo red stains, ubiquitin, and amyloid β protein immunostaining. We quantified the number of cerebral microbleeds, amount of iron, presence of amyloid β within the capillaries, and number of endothelial cells containing intranuclear inclusions. We evaluated the relationships between pathological findings using correlation analysis. We found intranuclear inclusions in the endothelial cells of capillaries and an increased number of cerebral microbleeds in the brains of those with fragile X-associated tremor/ataxia syndrome, both of which are indicators of cerebrovascular dysfunction. We also found a suggestive association between the amount of capillaries that contain amyloid β in the cerebral cortex and the rate of disease progression. We propose microangiopathy as a pathologic feature of fragile X-associated tremor/ataxia syndrome. © 2021 International Parkinson and Movement Disorder Society.

Cytopathology of bronchoalveolar lavages in COVID___19 pneumonia: A pilot study.

BACKGROUNDBronchoalveolar lavage (BAL) in patients with severe coronavirus disease 2019 (COVID‐19) may provide additional and complementary findings for the management of these patients admitted to intensive care units (ICUs). This study addresses the cytological features of the infection and highlights the more influential inflammatory components. The correlation between pathological variables and clinical data is also analyzed.METHODSThe authors performed a retrospective analysis of the cytopathological features of BAL in 20 COVID‐19 patients and 20 members of a matched cohort from a critical ICU who had acute respiratory distress syndrome caused by other pulmonary conditions.RESULTSA comparison of the controls (n = 20) and the COVID‐19 patients (n = 20) revealed that the latter had a higher neutrophil count (median, 63.8% of the cell count) with lower percentages of macrophages and lymphocytes. An increase in the expression of CD68‐positive, monocytic multinucleated giant cells (MGCs) was reported; megakaryocytes were not detected on CD61 staining. Perls staining showed isolated elements. In situ RNA analysis demonstrated scattered chromogenic signals in type II pneumocytes. An ultrastructural analysis confirmed the presence of intracytoplasmic vacuoles containing rounded structures measuring 140 nm in diameter (putative viral particles). In COVID‐19 patients, the clinicopathological correlation revealed a positive correlation between lactate dehydrogenase values and MGCs (r = 0.54).CONCLUSIONSThe analysis of BAL samples might be implemented as a routine practice for the evaluation of COVID‐19 patients in ICUs in the appropriate clinical scenario. Additional studies using a larger sample size of patients who developed COVID‐19 during the second wave of the epidemic in the autumn of 2020 are needed to further support our findings.

Characteristic vacuolisation of granulocytic and erythroid precursors associated with VEXAS syndrome.

A 78-year-old man with Sweet’s syndrome was referred for progressive anaemia. The full blood count showed macrocytic non-regenerative anaemia (haemoglobin concentration 82 g/l, mean cell volume 107 fl, reticulocytes 34 × 109/l) without neutropenia or thrombocytopenia. Bone marrow examination (images, ×100 objective) showed isolated dyserythropoiesis with basophilic stippling (top left), nuclear budding (bottom left), giant erythroblasts with nuclear:cytoplasmic asynchrony (bottom left) and 22% ring sideroblasts (Perls stain, top centre), without blast excess. This result was indicative of a myelodysplastic syndrome with ring sideroblasts and single lineage dysplasia (MDS-RS-SLD). Moreover, large vacuoles were observed in 20% of granulocytic (top right and bottom centre) and 10% of erythroid precursors (bottom right) but not in lymphocytes. Next-generation sequencing (NGS) did not reveal any classical MDS mutations but identified a UBA1 (ubiquitin-activating enzyme 1) M41L mutation (variant allele frequency 71%).

Ferroportin‐Dependent Iron Homeostasis Protects against Oxidative Stress‐Induced Nucleus Pulposus Cell Ferroptosis and Ameliorates Intervertebral Disc Degeneration In Vivo

Ferroptosis is a specialized form of regulated cell death that is charactered by iron-dependent lethal lipid peroxidation, a process associated with multiple diseases. However, its role in the pathogenesis of intervertebral disc degeneration (IVDD) is rarely investigated. This study is aimed at investigating the role of ferroptosis in oxidative stress- (OS-) induced nucleus pulposus cell (NPC) decline and the pathogenesis of IVDD and determine the underlying regulatory mechanisms. We used tert-butyl hydroperoxide (TBHP) to simulate OS conditions around human NPCs. Flow cytometry and transmission electron microscopy were used to identify ferroptosis, while iron assay kit, Perl's staining, and western blotting were performed to assay the intracellular iron levels. A ferroportin- (FPN-) lentivirus and FPN-siRNA were constructed and used to explore the relationship between FPN, intracellular iron homeostasis, and ferroptosis. Furthermore, hinokitiol, a bioactive compound known to specifically resist OS and restore FPN function, was evaluated for its therapeutic role in IVDD both in vitro and in vivo. The results indicated that intercellular iron overload plays an essential role in TBHP-induced ferroptosis of human NPCs. Mechanistically, FPN dysregulation is responsible for intercellular iron overload under OS. The increase in nuclear translocation of metal-regulatory transcription factor 1 (MTF1) restored the function of FPN, abolished the intercellular iron overload, and protected cells against ferroptosis. Additionally, hinokitiol enhanced the nuclear translocation of MTF1 by suppressing the JNK pathway and ameliorated the progression of IVDD in vivo. Taken together, our results demonstrate that ferroptosis and FPN dysfunction are involved in the NPC depletion and the pathogenesis of IVDD under OS. To the best of our knowledge, this is the first study to demonstrate the protective role of FPN in ferroptosis of NPCs, suggesting its potential used as a novel therapeutic target against IVDD.

Iron overload is related to muscle wasting in patients with cachexia of gastric cancer: using quantitative proteome analysis.

The incidence of gastric cancer cachexia is high and the clinical management is poor, so the study aimed to clarify the mechanism of muscle wasting to better screen patients with gastric cancer cachexia. Gastric cancer patients undergoing radical gastrectomy were divided into cachexia with sarcopenia (CS, n = 13) and normal (N, n = 10) two groups. The possible mechanism of skeletal muscle reduction was explored through Tandem Mass Tag (TMT) technique, Perls staining, Western blot analysis and measurement of oxidative stress indicators. The preoperative weight, weight loss, body mass index, calorie intake and skeletal muscle index values of the CS group were significantly lower than those of the N group (P < 0.05). We identified 114 differentially expressed proteins (DEP) in the muscles of two groups using TMT analysis. Bioinformatics analysis of DEP revealed that ferritin, iron and oxidative stress may be related to skeletal muscle consumption. Following Perls staining and measurement iron concentration in skeletal muscles, we found that the iron in the muscles of the CS group was significantly increased, and at the same time, western blot analysis showed that the expression of ferritin in the CS group was significantly increased and regulated by hepcidin-ferroportin axis. Finally, the CS group showed increased oxidative stress and weakened antioxidant stress systems in the muscles compared with the N group when oxidative stress indicators were analyzed. In conclusion, iron overload may be related to muscle loss in patients with gastric cancer cachexia. Gastric cancer patients with elevated ferritin are more likely to have muscle wasting.

LSD1 Inhibition with IMG7289 (bomedemstat) Rebalances Megakaryopoiesis and Erythropoiesis in a Patient with MPN/MDS with Refractory Anemia with Ringed Sideroblast and Marked Thrombocytosis with JAK2 and SF3B1 Mutations

Lysine-specific demethylase-1 (LSD1) is a histone H3 K4 demethylase that plays a critical role in hematopoietic progenitor cell differentiation, in particular, the production of megakaryocytes and erythrocytes from their common classical bi-potent progenitor (MEP). LSD1 is recruited to chromatin by the transcription factor GFI1B to license the maturation of megakaryocyte-erythrocyte progenitor cells (MEPs). Anemia is a common and serious complication of primary myelofibrosis (PMF), which is often refractory to conventional therapy. We report here the effect of IMG-7289 (bomedemstat), an LSD1 inhibitor, on skewing the fate of MEPs favoring the production of red cells over platelets and producing a complete hematologic remission in a JAK2V617F-positive PMF patient who had developed a refractory anemia with ringed sideroblasts. A 63 year old woman presented in 2016 with a 1 year history of with fatigue significantly compromising her lifestyle. She denied fever, night sweats, or weight loss. The physical examination was unremarkable with no palpable splenomegaly. Laboratory studies showed a hemoglobin (Hb) of 11.3 g/dL, a total white blood cell count (WBC) of 20.3k/µL with leukoerythroblastosis with 1% circulating blast cells, and a platelet count of 1,192k/µL. A bone marrow biopsy (BMB) showed a hypercellular marrow with increased dysmorphic megakaryocytes but without dyserythropoiesis. There were no ringed sideroblasts with Perls stain, and silver staining showed grade 2 reticulin fibrosis. The karyotype was del(7)(q11.2) and genotyping identified a JAK2V716F allele (VAF not known). The patient was considered to have PMF. Initial treatment consisted of hydroxyurea but without any improvement in her symptoms. Financial constraints initially prevented access to ruxolitinib but the patient was eventually started on 15 mg BID in January 2018. She tolerated therapy well but discontinued therapy a year later owing to no improvement in her fatigue and opted for observation alone. Over the next year, her hemoglobin fell to the range of 9.0-9.5g/dl and she developed drenching night sweats. A BMB in August 2019 showed a hypercellular marrow with increased dysplastic megakaryocytes but now with 76% ringed sideroblasts. Reticulin stain showed grade 2-3 fibrosis. No other dysplastic features were observed. Additional genotyping was declined by insurance and the patient was considered to have an MPN/MDS overlap syndrome. Accordingly, the patient was enrolled in clinical trial IMG-7289-CTP-102 (NCT03136185), a study of the LSD1 IMG-7289. On entry, her Hb was 9.5 gm/dL with an absolute reticulocyte count of 129K/µL. The WBC was 22.7k/µL with 69% neutrophils and 11% monocytes. The platelet count was 1,585k/µL with a mean platelet volume (MPV) of 8.1 fL. Genotyping showed a JAK2V617F VAF of 27%, a new SF3B1K700E mutation with a VAF of 26%, and a DNMT3A mutation with a VAF of 95%. Spleen volume by imaging was 283.3 cm³. The starting IMG-7289 dose was 40 mg po QD. At week 12, the Hb rose to 13.8 g/dL while the absolute reticulocyte count fell to 40K/µL. The platelet count was 271k/µL with the MPV increasing to 10.5 fL. The WBC was 5.2k/µL with 58% neutrophils and 18% monocytes. The mutant JAK2 allele burden had dropped to 14%. The spleen volume was 223 cm³. The IMG-7289 dose was reduced to 35 mg QD after complaints of fatigue. At Week 24, Hb was 11.7 g/dL, WBC was 12.1k/µL and platelets were 743k/µL. The total symptom score (MPN SAF TSS) was reduced by 31% from baseline. The expected pharmacodynamic effects of LSD1 inhibition on hematopoiesis were evident in this patient: monocytosis with a decrease in neutrophils and a marked reduction in the platelet count. Most striking was the concurrent improvement in the patient's anemia which was associated with a persistent reticulocytosis present at the start of treatment but with a significant increase in red blood cells in the setting of a SF3B1 mutation. MEP fate decisions hinge on the balance between KLF1 and FLI1 with the former favoring an erythroid fate, the later, megakaryocytes. The transcription factor complex of FLI1, GATA1, FOG1 which recruits the LSD1-containing NuRD complex is essential for megakaryocyte function; IMG-7289 likely disrupts that complex favoring erythropoiesis over megakaryopoiesis. The inhibition of LSD1 may, therefore, in the setting of thrombocytosis and anemia, rebalance the fate of MEPs to ameliorate both abnormalities. Disclosures Yacoub: Novartis: Speakers Bureau; Agios: Honoraria, Speakers Bureau; Incyte: Speakers Bureau; Hylapharm: Current equity holder in private company; Cara Therapeutics: Current equity holder in publicly-traded company; Ardelyx: Current equity holder in publicly-traded company; Dynavax: Current equity holder in publicly-traded company; Roche: Other: Support of parent study and funding of editorial support. Rienhoff:Imago BioSciences: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties.

Iron and inflammation: in vivo and post-mortem studies in Parkinson's disease.

In these present studies, in vivo and and post-mortem studies have investigated the association between iron and inflammation. Early-stage Parkinson's disease (PD) patients, of less than 5years disease duration, showed associations of plasmatic ferritin concentrations with both proinflammatory cytokine interleukin-6 and hepcidin, a regulator of iron metabolism as well as clinical measures. In addition ratios of plasmatic ferritin and iron accumulation in deep grey matter nuclei assessed with relaxometry T2* inversely correlated with disease severity and duration of PD. On the hand, post-mortem material of the substantia nigra compacta (SNc) divided according to Braak and Braak scores, III-IV and V-VI staging, exhibited comparable microgliosis, with a variety of phenotypes present. There was an association between the intensity of microgliosis and iron accumulation as assayed by Perl's staining in the SNc sections. In conclusion, markers of inflammation and iron metabolism in both systemic and brain systems are closely linked in PD, thus offering a potential biomarker for progression of the disease.

A case of hyperpigmentation induced by hydroxychloroquine and quinacrine in a patient with systemic lupus erythematosus and review of the literature.

Hydroxychloroquine (HQ) and quinacrine are widely used antimalarials for systemic lupus erythematosus (SLE) and other autoimmune diseases. We report a case of antimalarial-induced hyperpigmentation in a 57-year old African-American woman. The patient had a long-standing history of SLE that was treated with HQ and quinacrine in varying doses for 16 years. The disease improved considerably and entered remission; however, the patient subsequently developed severe, disseminated hyperpigmentation on her face, trunk, upper and lower extremities. A malar facial biopsy revealed numerous perivascular and scattered interstitial, heavily pigment laden dark brown to black macrophages. The Fontana Masson staining was positive for melanin, and Perl’s stain for iron was negative. This staining pattern, to our knowledge, has only been reported once before in the literature. Our patient represents a rare case of severe recalcitrant hyperpigmentation induced by combination HQ and quinacrine therapy for SLE.

Brainstem iron overload and injury in a rat model of brainstem hemorrhage

Background and PurposeBrainstem hemorrhage (BSH) is the most devastating subtype of intracerebral hemorrhage (ICH) with the highest mortality ranging from 56 % to 61.2 %. However, there is no effective medical or surgical therapy to improve its outcomes in clinic to date due to lack of understanding of its injury mechanisms. Herein, we explored the brainstem iron overload and injury in a rat model of BSH. MethodsNeurological scores were examined on day 1, 3, and 7 after modeling, and mortality of the rats was recorded to draft a survival curve. Rats were monitored by MRI using T2 and susceptibility weighted imaging (SWI) before sacrifice for examination of histology and immunofluorescence on day 1, 3, and 7. ResultsBSH rats had a high mortality of 56 % and demonstrated the severe neurological deficits mimicking the clinical conditions. SWI showed that the same increasing tendency in change of hypointense area with that in iron deposition by Perls staining from day 1 to 7. Expression of heme oxygenase 1 (HO-1) and generation of reactive oxygen species (ROS) had similar tendency and both peaked on day 3. Neuronal degeneration occurred and stayed elevated from day 1 to 7, while myelin sheath injury was initially observed on day 1 but without significant difference within 7 days. Conclusions: The time courses of erythrocyte lysis, HO-1 expression, iron deposition and ROS generation are related to each other after BSH. Besides, brainstem injury including neuronal degeneration and myelin damage were observed and discussed.

“Pauci-Hemosiderotic” Fibrolipomatous Tumor: A Mimicker of Various Lipomatous Lesions

Hemosiderotic fibrolipomatous tumor is a rare soft tissue tumor that preferentially affects the dorsum of foot, shows recurrent t(1;10) translocation targeting TGFBR3 and OGA (MGEA5) genes, and has a high recurrence potential. Hemosiderin deposits, mature adipocytes, and interspersed spindle cells are the 3 cardinal morphologic features of this tumor. We describe a "pauci-hemosiderotic" example involving the left wrist of a 45-year-old female, posing a diagnostic pitfall. The tumor comprised mature adipose tissue traversed by variably thick fibrous septa containing short fascicles of spindle cells. Prominent small- to medium-sized blood vessels were present, often with perivascular fibrosis or aggregates of foamy histiocytes, sometimes associated with red cell extravasation. Hemosiderin was not conspicuous, but fine deposits could be found focally on careful search and with the aid of Perls stain. The diagnosis was further confirmed by diffuse expression of CD34 and presence of OGA translocation by fluorescence in situ hybridization. Pathologists should be aware that hemosiderin deposition can be scanty and focal in hemosiderotic fibrolipomatous, but the rich vasculature with a "damaged" appearance is a useful diagnostic clue.

The Protective Effects of Cryptochlorogenic Acid on β-Cells Function in Diabetes in vivo and vitro via Inhibition of Ferroptosis.

PurposeMulberry leaf extract has exerted better antidiabetic activities, while the effects of major active components in mulberry leaf extract are still unclear. Cryptochlorogenic acid (CCA) as the major active component in mulberry leaf extracts was investigated herein.Materials and MethodsRats were treated with 50mg/kg streptozotocin for the establishment of diabetic model in vivo, and cells were treated with 33.3 mM glucose for the establishment of cell model in vitro. HE staining assay was performed for observation of pancreatic pathology and aldehyde fuchsin staining assay for examining islet cell numbers. The iron content was detected via Perls staining assay with iron assay kit (ab83366). The malondialdehyde (MDA), glutathione (GSH) and oxidized glutathione (GSSG) were detected by corresponding kits. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed for assessment of gene level and Western blot for measurement of protein expression level. The cell survival was detected via CCK-8 assay.ResultsThe blood glucose level, iron content, accumulation of lipid peroxides and islet injury in diabetic model were all improved by CCA via a concentration-dependent manner. CCA functions via inhibition of ferroptosis by activation of cystine/glutamate transporter system (XC−)/glutathione peroxidase 4(GPX4)/Nrf2 and inhibition of nuclear receptor coactivator 4 (NCOA4) in diabetes.ConclusionCCA exerted excellent antidiabetic effects via inhibition of ferroptosis, so it may be a promising agent for diabetes therapy, providing a new avenue for diabetes treatment.

Sevoflurane anesthesia during pregnancy in mice induces cognitive impairment in the offspring by causing iron deficiency and inhibiting myelinogenesis

Maternal anesthetic exposure during pregnancy is associated with an increased risk of cognitive impairment in offspring. The balance of cerebral iron metabolism is essential for the development of brain tissue. Iron deficiency affects the myelinogenesis and nerve tissue development, especially in fetus or infant, which has a key role in cognitive function. We aimed to investigate whether maternal sevoflurane (Sev) exposure caused cognitive impairment in offspring through inducing iron deficiency and inhibiting myelinogenesis. Pregnant mice (gestation stage day 14) were treated with 2% Sev for 6 h. Cognitive function of offspring mice was determined by the Morris water maze and Context fear conditioning test. Iron levels were assayed by Perl's iron staining and synchrotron imaging. Hippocampus and cortex tissues or cerebral microvascular endothelial cells of offspring mice (postnatal day 35) were harvested and subjected to Western blot and/or immunhistochemistry to assess ferritin, transferrin receptor 1(TfR1), Ferroportin-1 (FpN1), myelin basic protein (MBP), tight junction protein ZO-1, occludin, and claudin-5 levels. Beginning with postnatal day 30, the offspring were treated with iron therapy for 30 days, and the indicators above were tested. Our results showed Sev dramatically decreased the iron levels of brain and impaired cognitive function in offspring mice. Sev decreased the expression of heavy chain ferritin (FtH), light chain ferritin (FtL), MBP, ZO-1, occludin, claudin-5, and FpN1, and increased TfR1 in hippocampus and cortex or cerebral microvascular endothelial cells of offspring mice, indicating that Sev caused the iron deficiency and impaired the myelinogenesis in the brain of offspring. Interestingly, iron therapy prompted the myelinogenesis and improved impaired cognitive function at postnatal day 60. Our research uncovered a new mechanism which showed that iron deficiency induced by Sev and myelin formation disorder due to decreased iron of brain may be an important risk factor for cognitive impairment in offspring. It was necessary for offspring to be supplied iron supplement whose mother suffered exposure to sevoflurane during pregnancy.

Diagnosing Absolute Iron-Deficiency Anemia in Patients on Hemodialysis in a Tertiary Care Centre: A Retrospective Chart Review

Introduction: The gold standard for the diagnosis of absolute iron-deficiency anemia (IDA) in hemodialysis patients is a bone marrow aspirate with iron staining. Many clinicians use peripheral iron indices instead because they are non-invasive. Previous studies suggested that a serum ferritin &lt; 200 ng/mL was a reliable indicator of absolute iron deficiency in the hemodialysis population. However, the sensitivity of serum ferritin for the diagnosis of IDA in hemodialysis patients is poor. Methods: The primary objective of this study was to identify the optimal ferritin value to diagnose patients with absolute iron deficiency, as assessed on bone marrow aspiration, in the renal dialysis population. Secondary endpoints included the rate of clinically relevant findings on gastrointestinal investigation according to iron status. Research Ethics Board approval was obtained from Queen's University for this retrospective chart review. Hematopathology laboratory records were used to determine all individuals who had bone marrow examination at Kingston Health Sciences tertiary referral center between 2008 January 1 and 2018 August 21. This list was cross-referenced with the Nephrology dialysis database to identify the pre-specified study cohort; those who were receiving concurrent hemodialysis or peritoneal dialysis. Iron deficiency was defined as reduced or absent iron stores on bone marrow aspirate with Perl's Prussian blue stain. Anemia was defined as hemoglobin &lt;130 g/L in males and &lt;120 g/L in females. Additional parameters collected included ferritin (normal range 22 - 275 ng/mL male and 4 - 205 ng/mL female), transferrin saturation (TSAT, normal range 20-55%), vitamin B12, folate, albumin, CRP and thyroid function tests. Peripheral iron indices over six months were analyzed; statistical analysis was performed with t-tests and Mann-Whitney U tests. ROC curves were generated to determine the sensitivity and specificity of various threshold values for serum ferritin and TSAT. Results: Between 2008 January 1 and 2018 August 21, 4234 patients underwent bone marrow examination, of whom 28 had received renal dialysis replacement therapy at some point. Fifteen patients concurrently at the time of bone marrow testing receiving hemodialysis form the study population (Table 1). Among these fifteen patients, 6 (40%) were female, median age was 70.5 (range 39 - 80) years and all were anemic (Hb range 73 - 110 g/L). Four of these individuals were absolutely iron-deficient with reduced or absent iron stores by bone marrow evaluation. The mean ferritin and TSAT values for individuals with absolute iron deficiency by bone marrow aspiration was 273.5 ng/mL (n=4; median 224.5 ng/mL; range 158-539 ng/mL) and 22.0% (n=3; median 20%; range 20-26%), respectively. All four commenced erythropoietin stimulating agents; two received oral iron supplementation. Eight patients, including two of those determined to be absolutely iron deficient on bone marrow, had endoscopic investigation. Two were identified to have sources of gastrointestinal bleeding, both with ferritin values in the 100-200 range (106 and 189 ng/mL). With the limited sample size, the sensitivity and specificity of ferritin to identify absolute iron deficiency in this hemodialysis population was 50% and 85%, respectively, at a threshold of 198 ng/mL. Discussion: Ferritin and TSAT are not sensitive markers for absolute iron deficiency in hemodialysis patients. Bone marrow examination is performed in a minority. The small sample size in this study precludes definitive determination of an optimal ferritin cut-off to diagnose iron deficiency in the dialysis population. Uncertainty about actual iron status may result in alternative invasive testing, such as colonoscopy, to investigate the cause of their anemia. Newer tests such as reticulocyte hemoglobin content and percent hypochromic red blood cells are more accurate and may guide diagnosis and management of IDA in hemodialysis patients. However they are not always routinely available. Further studies are needed to compare the utility of these peripheral iron indices to the gold standard bone marrow examination in a larger population, to allow identification of patients with absolute or functional IDA, and minimize invasive and potentially unnecessary investigation. Disclosures Hay: AbbVie: Research Funding; Kite: Research Funding; Janssen: Research Funding; Seattle Genetics: Research Funding; Celgene: Research Funding; MorphoSys: Research Funding; Roche: Research Funding; Novartis: Research Funding; Gilead: Research Funding; Takeda: Research Funding.

Gas6 Targeting Promotes Major Bleeding and Aggravates Inflammation in Protein S Deficient Mice

Background: Protein S (PS) and growth arrest-specific gene 6 (Gas6) are vitamin K-dependent proteins. PS is an anticoagulant whose pivotal role is illustrated by purpura fulminans (PF), a life-threatening condition characterized by purpuric skin lesions, disseminated intravascular coagulation and thrombosis. Unlike PS, Gas6 displays a procoagulant effect. Its complete deficiency has anti-hemostatic, pro-inflammatory and anti-erythropoietic effects. In tissues, PS and Gas6 have an impact on cellular functions by binding to and activating receptor tyrosine kinases of the TAM family. PS complete deficiency-induced PF is thought to result from the imbalance between procoagulant and anticoagulant forces. However, its pathophysiological mechanism has not yet been fully elucidated. Previous studies showed that Pros1-/-mice die in utero with a PF phenotype similar to the one observed in human. We therefore considered Pros1-/- mice as a suitable model of the human PF. The aim of this work was to investigate if Gas6 deficiency could rebalance hemostasis in PS deficient mice and rescue them from PF. Methods &amp; results: Pros1 +/- Gas6 -/- mice were intercrossed to obtain Pros1-/-Gas6-/- mice. Embryonic mortality was higher in Pros1+/-Gas6-/- than in Pros1+/- mattings (6% vs 3 % at E14, 21% vs 18 % at E16, 30% vs 20 % at E17, respectively). About 7.5% of dead embryos from Pros1+/-Gas6-/- mattings were found necrotic and macerated versus only 3.8% inPros1+/- mattings. Macroscopically, 74% of Pros1-/-Gas6-/- embryos displayed maximal bleeding scores (3) corresponding to intracranial and major body bleedings compared to 47% in Pros1-/- embryos (p&amp;lt;0.02). Microscopically, all Pros1-/-Gas6-/- embryos had a bleeding score of 3, whereas 50% of Pros1-/- embryos displayed a milder bleeding score (2). Major blood vessels of E16 Pros1-/- and Pros1-/-Gas6-/- embryos contained a high number of non-phagocyted red blood cells (RBC) expelled nuclei (38% and 58%, respectively) while only few of them were found in Pros1+/+Gas6+/+ embryonic vessels (5%), indicating that altered phagocytosis due to the lack of PS was further aggravated by Gas6 deficiency (p&amp;lt;0.003). More immature RBC were found in Pros1-/- than in Pros1+/+Gas6+/+ circulating blood (17% vs 12%, p&amp;lt;0.004), suggesting increased erythropoiesis in Pros1-/- embryos in response to bleeding. These data were confirmed using FACS analysis of CD71/Ter119 stained liver single cell suspensions. E14 Pros1-/- embryos had more immature RBC compared to Pros1+/+Gas6+/+ embryos (2.3% vs 1.4%, p&amp;lt;0.08). However, Pros1-/-Gas6-/- embryos displayed less immature RBC in their circulating blood than Pros1-/- embryos. The number of erythroid burst-forming units was lower in Pros1-/-Gas6-/- fetal livers compared with Pros1-/- fetal livers (5±1.3 vs 8±2, p&amp;lt;0.08) confirming reduced erythropoiesis in Pros1-/-Gas6-/- embryos. Perls staining showed increased iron stores in fetal liver and numerous intra-vascular hypochromic RBC in Pros1-/-Gas6-/- embryos pointing to a functional iron deficiency. We also investigated E15 embryonic dorsal skin by immunofluorescence. We found RBC extravasation (VE-cadherin and Ter119 stainings), vascular network outgrowth impairment with fewer vessel branches (CD31 staining), massively enlarged lymphatic vessels and increased macrophages infiltration (Lyve1 and F4/80 stainings) in Pros1-/-Gas6-/- and Pros1-/- indicating an ongoing inflammatory process. Conclusion: Gas6 deficiency did not rebalance hemostasis in Pros1-/-embryos and prevent PF. Notably, Gas6 and PS combined deficiency leads to a more dramatic phenotype than PS deficiency alone with a higher mortality rate, more major bleedings, phagocytosis impairment, more inflammation and an erythropoietic defect compatible with anemia of inflammation. Disclosures No relevant conflicts of interest to declare.

Assessment and grading of pigmentation in chronic venous insufficiency.

Chronic venous insufficiency causes skin pigmentation of the leg ranging from small patches of mild dyschromia to extensive areas of severe skin pigmentation. It is thought that the pigmentation is mainly due to haemosiderin or melanin deposition. Erythrodiapedesis which occurs as a result of venular hypertension causes erythrocytes to migrate across the microvascular network into the dermis. We categorized the grading of pigmentation into four grades: +, few spots; ++, pigmentation over gaiter area; +++, pigmentation involving leg and ankle; ++++, heavily pigmented (dark). Skin biopsies were taken from the patient while undergoing surgery; two biopsies were taken from each patient, one from apparently normal skin and other from the site of pigmentation. A total of 45 patients diagnosed as chronic venous insufficiency with pigmentation were included in the study and five patients included in control. The biopsy specimens were sent to pathology department for H&E, Perls stain and IHC for S100. Majority of cases, i.e. 62% of limbs fall under (++) grade of pigmentation, followed by (+) grade of pigmentation in 20%, while (+++) and (++++) constitute 9% of the cases each. Increased melanin deposition was seen in 40 pigmented skin biopsies and 3 normal skin biopsies from the case group, and normal melanin deposition was seen in all the non-varicose controls. We have tried to categorize pigmentation in chronic venous insufficiency into four grades. As the grade of pigmentation increases the per cent of cases with ulceration is increasing. It was observed that presence of melanin deposition irrespective of the grade of pigmentation was distributed more towards the advanced clinical classification (C5 and C6).

Quantitative Iron Neuroimaging Can Be Used to Assess the Effects of Minocycline in an Intracerebral Hemorrhage Minipig Model.

Iron-mediated toxicity is a key factor causing brain injury after intracerebral hemorrhage (ICH). This study was performed to investigate the noninvasive neuroimaging method for quantifying brain iron content using a minipig ICH model and assess the effects of minocycline treatment on ICH-induced iron overload and brain injury. The minipig ICH model was established by injecting 2ml of autologous blood into the right basal ganglia, which were then subjected to the treatments of minocycline and vehicle. Furthermore, the quantitative susceptibility mapping (QSM) was used to quantify iron content, and diffusion tensor imaging (DTI) was performed to evaluate white matter tract. Additionally, we also performed immunohistochemistry, Western blot, iron assay, Perl's staining, brain water content, and neurological score to evaluate the iron overload and brain injury. Interestingly, we found that the ICH-induced iron overload could be accurately quantified by the QSM. Moreover, the minocycline was quite beneficial for protecting brain injury by reducing the lesion volume and brain edema, preventing brain iron accumulation, downsizing ventricle enlargement, and alleviating white matter injury and neurological deficits. In summary, we suggest that the QSM be an accurate and noninvasive method for quantifying brain iron level, and the minocycline may be a promising therapeutic agent for patients with ICH.

Blood Cells: Morphology and Clinical Relevance, 2nd edn, G.Gulati and J.Caro, ASCP Press, Chicago, 2019. ISBN 978‐089189‐6234

This book is an annotated atlas, which will have a useful role in diagnostic haematology laboratories. The images are of high quality and are generally taken at a consistent magnification so that they are readily comparable with each other. The book is very up-to-date incorporating, for example, the 2016 World Health Organization Classification of Tumours of Haematopoietic and Lymphoid Tissues. A self-assessment chapter is a useful inclusion, which could be first used either before or after reading the book, depending on the degree of experience of the reader. The annotations of the images are based on the authors’ considerable experience and no references are cited. Occasionally a reference would have been appreciated; for example, what is the evidence that the in vitro phenomenon of platelet satellitism is associated with an increased incidence of thromboembolism? The level of accuracy is high, although I did find two figures with their legends reversed and it is Perls stain not Perl stain. What might be added to a further edition? Circulating tumour cells are mentioned but not illustrated and I could not find a description of South-East Asian ovalocytosis or of the distinctive features of paroxysmal cold haemoglobinuria (the combination of spherocytes, small agglutinates and erythrophagocytosis). But these are mere quibbles. Haematologists and biomedical scientists will appreciate having this book on their laboratory shelves.

Complement C3 participates in the function and mechanism of traumatic brain injury at simulated high altitude

BackgroundTraumatic brain injury (TBI) leads to severe mortality and disability, in which secondary injury induced by complement activation plays an important role. TBI tends to be associated with more severe cerebral edema and worse neurological functional recovery if it occurs in high-altitude areas than in low-altitude areas. However, the underlying mechanism of this difference is unknown. Thus, we used cobra venom factor (CVF) to deplete complement C3 in simulated high-altitude areas to explore whether the differences in outcome at different altitudes are related to secondary injury caused by complement C3. MethodsThe weight-drop model was adopted to induce TBI in rats. Rats were randomly divided into the following groups: sham + saline (sham), high altitude + TBI + saline (HAT), high altitude + TBI + CVF (H-CVF), low altitude + TBI + saline (LAT), and low altitude + TBI + CVF (L-CVF). Brain contusion and edema volumes, brain water content, myelin basic protein (MBP) expression, tumor necrosis factor alpha (TNF-a) expression, interleukin 1 beta (IL1B) expression, mortality rate, neurological function, and complement component 3 (C3) mRNA expression were measured by techniques such as Evans blue fluorescence, Perls staining, TUNEL staining, ELISA, immunohistochemistry and Western blotting to evaluate correlations between complement activation and secondary injury. ResultsThe activation of complement after TBI was significantly higher at high altitude than at low altitude. High-altitude TBI resulted in a leakier blood-brain barrier, more severe cerebral edema and higher mortality than low-altitude TBI did. In addition, high-altitude TBI tended to be associated with more MBP degradation, ferric iron deposition, neuronal apoptosis, and inflammatory factor deposition than low-altitude TBI. All of these effects of TBI were partially reversed by inhibiting complement activation using CVF. ConclusionOur study provided evidence that TBI at high altitude leads to severe edema and high mortality and disability rates. Complement C3 activation is one of the important factors contributing to secondary brain injury.

5-Aza-2′-deoxycytidine increases hypoxia tolerance-dependent autophagy in mouse neuronal cells by initiating the TSC1/mTOR pathway

BackgroundOur previous study found that 5-Aza-2′-deoxycytidine (5-Aza-CdR) can repress the expression and activity of protein serine/threonine phosphatase-1γ (PP1γ) in mouse hippocampus. It is well known that PP1γ regulates cell metabolism, which is related to hypoxia/ischaemia tolerance. It has been reported that it can also induce autophagy in cancer cells. Autophagy is important for maintaining cellular homeostasis associated with metabolism. In this study, we examined whether 5-Aza-CdR increases hypoxia tolerance-dependent autophagy by initiating the TSC1/mTOR/autophagy signalling pathway in neuronal cells. Methods5-Aza-CdR was either administered to mice via intracerebroventricular injection (i.c.v) or added to cultured hippocampal-derived neuronal cell line (HT22 cell) in the medium for cell culture. The hypoxia tolerance of mice was measured by hypoxia tolerance time and Perl's iron stain. The mRNA and protein expression levels of tuberous sclerosis complex 1 (TSC1), mammalian target of rapamycin (mTOR) and autophagy marker light chain 3 (LC3) were measured by real-time PCR and western blot. The p-mTOR and p-p70S6k proteins were used as markers for mTOR activity. In addition, the role of autophagy was determined by correlating its intensity with hypoxia tolerance in a time-dependent manner. At the same time, the involvement of the TSC1/mTOR pathway in autophagy was also examined through transfection with TSC1 (hamartin) plasmid. Results5-Aza-CdR was revealed to increase hypoxia tolerance and induce autophagy, accompanied by an increase in mRNA and protein expression levels of TSC1, reduction in p-mTOR (Ser2448) and p-p70S6k (Thr389) protein levels, and an increase in the ratio of LC3-II/LC3-I in both mouse hippocampus and hippocampal-derived neuronal cell line (HT22). The fluorescence intensity of hamartin was enhanced in the hippocampus of mice exposed to 5-Aza-CdR. Moreover, HT22 cells that over-expressed TSC1 showed more autophagy. Conclusions5-Aza-CdR can increase hypoxia tolerance by inducing autophagy by initiating the TSC1/mTOR pathway.