Marked Anemia Research Articles

A rare case of glanzmann thrombasthenia with concomitant factor VII deficiency.

Glanzmann thrombasthenia and clotting factor VII deficiency are rare autosomal recessive bleeding disorders. But the occurrence of both in the same person is an extremely rare phenomenon. Here, we present the case of a young female from Sindh, Pakistan that got diagnosed with Glanzmann thrombasthenia and concomitant moderate factor VII deficiency, a combination not previously reported in the country. The patient exhibited typical clinical manifestations including menorrhagia, nasal bleeds, and prolonged bleeding after minor injuries, compounded by a positive family history and consanguinity. Laboratory investigations revealed marked anemia, prolonged bleeding time, and abnormal platelet aggregation studies consistent with Glanzmann thrombasthenia. The identification of this rare combination relied on comprehensive clinical evaluation, emphasizing the importance of family history in suspected cases. Management involved platelet transfusions, tranexamic acid, and Factor VII replacement, resulting in clinical improvement.

Lymphomas with massive splenomegaly – a case series

Hairy cell leukaemia (HCL) is an uncommon chronic B-cell lymphoma. Classical HCL and its variant are separate entities. Splenic marginal zone lymphoma (SMZL) is rare accounting for <2%of lymphoid neoplasms2. A 62-year-old woman presented with constitutional symptoms and mild splenomegaly was having pancytopenia, monocytopenia and hairy cells in the peripheral blood. Bone marrow aspiration was a dry tap while trephine biopsy revealed diffuse lymphoid cell infiltration with characteristic fried egg appearance and fibrosis. Morphological and immunophenotypic diagnosis was HCL. She was successfully treated with IV-cladribine and G-CSF for chemotherapy induced severe neutropenia. She achieved complete remission in 6 months. A 56-year-old woman presented with abdominal distension and lower limb swelling was having marked splenomegaly, absolute lymphocytosis, mild neutropenia and anaemia. Blood film revealed lymphocytes showing moderate cytoplasm and cytoplasmic projections. With bone marrow biopsy and flow cytometry, splenic B-cell lymphoma/leukaemia with prominent nucleoli was diagnosed and was treated with rituximab monotherapy. A 76-year-old woman presented with progressive abdominal fullness, unintentional weight loss and early satiety was having splenomegaly, bicytopenia and absolute lymphocytosis consisting predominantly of small mature lymphocytes and occasional lymphocytes with polar villi. Morphological and immunopheno-typical results confirmed SMZL. She was treated with rituximab monotherapy.

Therapeutic Management of Canine Ehrlichiosis: Lesson Learned from Bareilly, India

The study was conducted on 5 dogs presented at Teaching Veterinary Clinical Complex, Indian Veterinary Research Institute with history of anorexia, lethargy, weakness, high fever, vomiting, severe panting and heavy tick infestation. Physical examination of dogs revealed increased respiratory rate, pale mucus membrane and conjunctiva, dehydration and enlarged lymph nodes. Haematological finding showed marked anaemia and thrombocytopenia. Sonographic and radiographic investigation of the abdomen revealed splenomegaly and partial hepatomegaly. Parasitological examination found it positive for Ehrlichia canis and confirmed the ticks Rhipicephalus sanguineus. Dog was treated with doxycycline @ 10 mg/kg for 28 days in divided dose with supportive therapy. Dog showed marked recovery after 2 dose of doxycycline and re-examination of blood after 20 days gave negative results in rapid test kit.

The Effect of Repeated Blood Harvesting from Pregnant Mares on Haematological Variables.

Studies have been carried out on the effect of large-volume blood harvesting from horses, but they were performed on nonpregnant horses of various breeds other than Icelandic horses. This study aimed to investigate the effect on the haematological variables indicating erythropoiesis of repeated blood harvesting from pregnant mares. To account for regional variation, two herds of mares were chosen, both kept under free-range conditions. Sequential EDTA blood samples were collected weekly from 160 mares and analysed for haematological variables in an automated analyser. Serum samples from 115 mares were analysed for total protein. In both herds, after three harvests, mares began to measure below the minimum value for erythrocyte numbers, and mild anaemia was present in up to 37% at one time. Mares in only one herd had moderate or marked anaemia, 14.3% of the herd. Both herds showed evidence of increased erythropoiesis, but there was a difference between the herds in the intensity of the response. In both herds, however, all mares had reached the minimum normal haematocrit value three weeks after the last harvest. It is important to investigate the causes for the differences between these herds, which might be addressed to reduce the risk of anaemia.

Icterícia em gatos: causas e resultados post-mortem em 44 casos

ABSTRACT: The aim of this study was to describe the pathological findings and causes of jaundice in 44 cats (Felis catus) over a six-year period. The cats were from two Brazilian metropolitan areas: 34.1% were female, 56.8% were male, and 9.1% had no information regarding their sex. Their ages ranged from 6 months to 13 years. Most of the cats examined were of a mixed breed (40/44), whereas the others were Angora (2/44), Oriental Short Hair (1/44) and Persian (1/44). All animals had mild to marked jaundice, and 39 were diagnosed with mild to marked anemia. The classification of icterus types (pre-hepatic, hepatic and post-hepatic) was based on gross and microscopic findings. Of the 44 animals, 10 were classified as pre-hepatic icterus, 33 with hepatic icterus and seven with post-hepatic icterus. In some cats, two types of icterus were found, of which five were classified as pre-hepatic and hepatic icterus, and one case was hepatic and post-hepatic icterus. According to the gross and microscopic findings, the cause of pre-hepatic icterus was idiopathic hemolytic anemia. The most frequent cause of hepatic icterus was hepatic lipidosis (26/44), followed by perihepatitis and hepatitis compatible with feline infectious peritonitis, lymphoma, glycogenic degeneration, cholangiocarcinoma and metastatic myeloid leukemia. In animals with post-hepatic icterus, the causes included cholangitis due to Platynosomum spp. infection, cholangioma of the common hepatic duct, and chronic cholangitis. Understanding the etiopathogenesis of jaundice requires an accurate clinic-pathological study and concomitant causes of the disease in cats should be considered.

Complete Blood Count Values Over Time in Young Children During the Dengue Virus Epidemic in the Dominican Republic From 2018 to 2020.

Background: Dengue fever (DF) is a mosquito-borne illness with substantial economic and societal impact. Understanding laboratory trends of hospitalized Dominican Republic (DR) pediatric patients could help develop screening procedures in low-resourced settings. We sought to describe laboratory findings over time in DR children with DF and DF severity from 2018 to 2020. Methods: Clinical information was obtained prospectively from recruited children with DF. Complete blood count (CBC) laboratory measures were assessed across Days 1-10 of fever. Participants were classified as DF-negative and DF-positive and grouped by severity. We assessed associations of DF severity with demographics, clinical characteristics, and peripheral blood studies. Using linear mixed-models, we assessed if hematologic values/trajectories differed by DF status/severity. Results: A total of 597 of 1101 with a DF clinical diagnosis were serologically evaluated, and 574 (471 DF-positive) met inclusion criteria. In DF, platelet count and hemoglobin were higher on earlier days of fever (p < = 0.0017). Eighty had severe DF. Severe DF risk was associated with thrombocytopenia, intraillness anemia, and leukocytosis, differing by fever day (p < = 0.001). Conclusions: In a pediatric hospitalized DR cohort, we found marked anemia in late stages of severe DF, unlike the typically seen hemoconcentration. These findings, paired with clinical symptom changes over time, may help guide risk-stratified screenings for resource-limited settings.

Investigating the association between intestinal parasite infection and cribra orbitalia in the medieval population of Cambridge, UK

ObjectiveCribra orbitalia is believed to be a skeletal indicator of chronic anaemia, scurvy, rickets or related metabolic diseases. It has been suggested that it may be used as a proxy indicator for intestinal parasite infection, as parasites often cause anaemia today. Our aim is to investigate this association in the medieval population of Cambridge, UK. MaterialsIndividuals excavated from the cemeteries of the Augustinian friary and All Saints by the Castle parish church, and aged from 7 to adulthood. MethodsWe undertook parasite analysis of the pelvic sediment and control samples of 46 burials with intact orbital roofs. ResultsHuman roundworm (Ascaris lumbricoides) and/or whipworm (Trichuris trichiura) were identified in the pelvic sediment of 22 individuals, and cribra orbitalia noted in 11 individuals. Barnards test showed no association between parasite infection and cribra orbitalia (p = .882). ConclusionWe found no association between infection and cribra orbitalia infection in this medieval adult population, calling into question this hypothesis, at least for adults. SignificanceHigh or low cribra orbitalia prevalence in adults should not be used to infer rates of intestinal parasite infection. LimitationsThe individuals in the study were over the age of 7, with no younger children. It is possible that only parasites which cause marked anaemia (such as hookworm, schistosomiasis or malaria) may cause cribra orbitalia, while less marked anaemia from roundworm and whipworm may not do so. Suggestions for Further ResearchRepeating this study in younger children, when most cribra orbitalia appears to form.

A Sticky Situation: Diagnosing Post-Transfusion Purpura in the Setting of Longstanding, Transfusion-Dependent AML

Background Post-transfusion purpura (PTP), a commonly underreported adverse transfusion event. It is defined as severe thrombocytopenia with related sequelae occurring 5-10 days of transfusion where both transfused and autologous platelets are destroyed. Low cell counts may be masked by other conditions (i.e. AML) and there is a paucity of literature on cases of PTP especially in concurrent pancytopenia. Swift diagnosis is critical for management. This case presents a unique opportunity to examine PTP in a patient with a complex hematopathologic malignancy history. Methods Relevant clinical data was obtained through examination of available medical records, concurrent ancillary diagnostic testing, and serological send-out testing was performed. Appropriate investigation and evaluation of repeated transfusion reactions with clinical symptomatology in accordance with available bio-vigilance surveillance guidelines aided in the diagnosis. Findings A 73-year-old female patient presented to the emergency department with acute, worsening fatigue, lethargy, pallor, and shortness of breath. She was found at home by emergency medical services with bright red blood covering her legs, originating from the rectum, several longstanding pressure ulcers, and an oxygen saturation in the 80s. The initial physical exam revealed a cachectic, ill-appearing woman who was tachypneic, wheezing, with new and old blood at anus, generalized weakness, fatigue, shortness of breath, cough, myalgias, and diffuse bruising of various sizes. There was no loss of consciousness or history of anticoagulant therapy. The patient had a complex medical history which included acute myeloid leukemia with concurrent chemotherapy complicated by longstanding, transfusion-dependent pancytopenia, lung adenocarcinoma status-post radiation therapy now in remission, congestive heart failure, chronic obstructive pulmonary disease on continuous home oxygen, chronic intermittent bilateral lower extremity rashes secondary to adult-onset Still's disease, and multiple visits to the emergency department, inpatient admissions, and numerous outpatient infusions for the management of pancytopenia. The patient's initial laboratory workup in the emergency room setting demonstrated clear, previously known, pancytopenia along with severe acidosis, marked anemia, and derangements of coagulation testing. With numerous visits to both the emergency department and outpatient infusion centers, the patient had previous transfusion reactions which ranged from mild allergic to repeating patterns of fulminant weakness, lethargy, and confusion relating to the timing of transfusions. With increasing skin manifestations of purpuric skin lesions, severely acute thrombocytopenia regardless of HLA matched or compatible platelet units, and results of send-out serology demonstrating 100% reactivity to all platelets and specific HPA antibodies, the diagnosis of post-transfusion purpura was made. Conclusion Despite this patient succumbing to diffuse hypoxic anemia and suffering a fatal cardiac dysrhythmia, the results of reference laboratory send-out serology revealed positivity in 100% of platelets crossmatched and the presence of antibodies to HLA Class I and HPA-2b, which satisfy the clinical and serologic criteria for post-transfusion purpura and represent a rare diagnostic finding.

A Rare Case of Visceral Leishmaniasis Diagnosed by Endoscopy in an Anemic Patient With HIV/AIDS

ABSTRACT Visceral leishmaniasis (VL) is a rare protozoan parasitic disease transmitted by sandfly vectors predominantly found in Asia and Central America. Although far less common than cutaneous leishmaniasis, VL carries a higher mortality burden because of its propensity to cause emaciation and marked anemia through bone marrow infiltration. Treatment is particularly challenging in patients coinfected with HIV before both infections work synergistically to perpetuate immunosuppression, further promoting higher viral loads and disease burden. In comparison with those without HIV, these patients have poorer treatment responses and higher rates of relapse. We describe a case of VL in an anemic patient with HIV/AIDS diagnosed during esophagogastroduodenoscopy. We also present challenges to treatment, adverse drug side effects, and long-term sequelae.

Anemia in patients with necrotizing soft tissue infections, pathogenetic and prognostic value

BACKGROUND: Necrotizing soft tissue infection is one of the most severe life-threatening surgical infections with a very high mortality rate. A characteristic feature of necrotizing soft tissue infection is the rapid development of anemia, the causes and prognostic value of which are not well understood.&#x0D; AIM: The purpose of this study was to investigate the timing, development, and dynamics of anemia in generalized forms of necrotizing infection to identify clinical and bacteriological factors associated with its development.&#x0D; MATERIALS AND METHODS: 129 patients with necrotizing soft tissue infection who were treated from 09.2015 to 12.2019 in the department of purulent-septic surgery at Hospital of the Holy Great Martyr George were examined. All patients received surgical treatment, laboratory hematological, biochemical examination, bacteriological examination of blood, and wound discharge. Overall, 22 patients suffered from systemic inflammatory response syndrome, 63 patients with sepsis, and 41 patients with septic shock.&#x0D; RESULTS: The Counts of hemoglobin and red blood cells in necrotizing soft tissue infection patients with sepsis revealed the anemia already during the first day and then from the 15th day of the disease, the red blood cell values began to rise in the patients who survived. However, continued to decrease in the deceased patients. In the group of deceased sepsis patients from day 3 of hospitalization, correlations between red blood cells count and potassium ion concentration (r = 0.318; p 0.01), and red blood cells count and total plasma protein (r = 0.30; p 0.01) became significant. Among patients with hemoglobin 110 g/L on the day of hospitalization, 36 of 67 (53.7%) patients died, and among those with hemoglobin levels 110 g/L, 20 of 62 (32.2%) patients died (p = 0.004). The highest lethality was registered patients who suffered from wound discharge Klebsiella pneumoniae (12 of 18, 66.7%) or anaerobic infection, but marked anemia was noted only in patients with anaerobic infection (Proteus spp., Clostridium spp., Bacteroide spp.) (8 out of 12, 66.7%).&#x0D; CONCLUSIONS: We attribute the development of anemia in sepsis patients to the destruction of red blood cells. The type of infectious agent influences both the mortality rate and the degree of anemia, which is probably related to the ability of bacteria to destroy red blood cells.

Misunderstood Gastric Perforation of a Pancreatic Acinar Cell Carcinoma: A Wolf in Sheep’s Clothing

A 70-year-old man was admitted to the Emergency Department (ED) for marked asthenia and severe anemia. In addition, a high level of lipase was found. During hospitalization, a locally advanced gastric cancer was diagnosed, with endoscopic evidence of a large polyploid formation originating under the cardias that occupied most of the gastric lumen. A total body CT scan was performed before surgery; the tumor affected the posterior gastric wall, with tenacious infiltration of the pancreatic body. Therefore, we performed a total gastrectomy with esophageal jejunum anastomosis and reconstruction of intestinal continuity according to Roux, distal spleno-pancreatectomy, and cholecystectomy. At histology, a pancreatic acinar cell carcinoma (PACC) with full thickness infiltration of the gastric wall was diagnosed. Acinar cell carcinomas are highly aggressive neoplasms, and surgical resection, when feasible, is the treatment of choice regardless of size, also because the role of neoadjuvant or adjuvant chemo- or radiotherapy remains uncodified.

Clonal Evolution of t(8;21) Coexisting with t(9;22) in a Blastic Transformation of Chronic Myeloid Leukemia

Abstract Introduction/Objective Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by chromosomal translocation t(9;22) and the production of the fusion protein BCR-ABL1. The clinical course of the disease is characterized by a chronic phase, accelerated phase, and blast phase. One of the criteria for the progress of chronic phase to accelerated phase is the presence of cytogenetic anomaly additional to t(9;22). The blast phase of CML usually presents as acute myelogenous leukemia (AML); however, its characteristics are different from de novo AML. One of the most common subtypes of de novo AML with balanced translocation is AML with t(8;21). Here, we present a case of the blast phase of CML that presents t(8;21). Methods/Case Report The peripheral blood from a 51-years-old-man presenting with fatigue and splenomegaly showed leukocytosis, mild anemia, marked neutrophilia with left shift, monocytosis, basophilia, and &amp;lt;1% blasts. Flow cytometry identified an increase in granulocyte events with the left shift. Chromosome studies detected t(9;22) by karyotyping and FISH. BCR-ABL1 transcript was detected at 41.98% on the International Scale by RT-PCR, consistent with chronic myeloid leukemia (CML). The following bone marrow biopsy confirmed a diagnosis of the chronic phase of CML with &amp;lt;5% myeloblasts. After a year of treatment with imatinib, the patient developed marked leukocytosis, anemia, and thrombocytopenia. Flow cytometry detected an immature cell population comprising ~50% of total cells positive for CD117, CD34 (partial), CD13 (dim), and CD33. A few blasts were also positive for cMPO and negative for CD14, CD64, TdT, Glycophorin A, cytoplasmic CD3, CD19, and other markers. BCR-ABL1 transcript was detected at 56.45% on the International Scale by RT-PCR. Chromosome analysis revealed all cells (20/20) exhibiting t(8;21) leading to a fusion between RUNX1 and RUNX1T1 genes with a maintained t(9;22). FISH analysis confirmed t(8;21) and t(9;22). These findings were consistent with a blast phase of CML. Results (if a Case Study enter NA) NA Conclusion Progression of CML is frequently accompanied by cytogenetic evolution. However, the development of t(8;21) in Ph+ clones is extremely rare. Only six cases have been previously reported. Misidentifying this blast phase of CML (in the absence of a clinical history of the chronic phase of CML) as AML with recurrent genetic abnormalities is a potential pitfall we should be aware of.

Virus and Autoimmunity: Can SARS-CoV-2 Trigger Large Vessel Vasculitis?

IntroductionViral infections can induce autoimmune diseases in susceptible patients. SARS-CoV-2 has been associated with the development of rheumatic disease, especially small vessel vasculitis and arthritis. Typically, onset occurs days to weeks after the antigenic challenge and in patients with mild COVID-19. We report a case of large vessel vasculitis (LVV) temporally related to SARS-CoV-2 infection.Case descriptionAn otherwise healthy 19-year-old woman presented with fatigue, malaise, and chest and low back pain. The symptoms had begun 5 weeks earlier and 1 month after mild SARS-CoV-2 infection. Serological work-up revealed a marked proinflammatory state and anaemia without signs of infectious or autoimmune disease. Computerized tomography revealed thickening and blurring of the perivascular fat of the descending thoracic and abdominal aorta, progressing along the proximal iliac and renal arteries. Fluorodeoxyglucose positron emission tomography confirmed inflammatory activity. Symptoms and laboratory values normalized after prednisolone treatment.DiscussionRecent SARS-CoV-2 infection may be a trigger for LVV, including Takayasu arteritis, as well as other rheumatic diseases. A prompt and thorough differential diagnosis is essential to exclude aortitis and LVV mimickers. Moreover, physicians should be aware of the potential spectrum of systemic and autoimmune diseases that could be precipitated by SARS-CoV-2 infection. This will allow timely diagnosis and treatment, with significant improvement in prognosis.LEARNING POINTSSARS-CoV-2 infection can trigger large vessel vasculitis and other rheumatic diseases.Awareness of the association between COVID-19 and autoimmune phenomena allows for timely diagnosis and treatment with significant improvements in prognosis.Vasculitis and other autoimmune diseases should be kept in mind in patients who develop proinflammatory states days to weeks after an initial antigenic challenge.

Kidney xenotransplantation in a brain-dead donor: Glass half-full or half-empty?

Kidney xenotransplantation in a brain-dead donor: Glass half-full or half-empty?

Low Frequency of Folate and Vitamin B12 Deficiency in Patients with Marked Macrocytic Anemia.

Low Frequency of Folate and Vitamin B12 Deficiency in Patients with Marked Macrocytic Anemia.

Short-Term Immune Responses of Gilthead Seabream (Sparus aurata) Juveniles against Photobacterium damselae subsp. piscicida.

Photobacteriosis is a septicaemic bacterial disease affecting several marine species around the globe, resulting in significant economic losses. Although many studies have been performed related to the pathogen virulence and resistance factors, information regarding the host defence mechanisms activated once an infection takes place is still scarce. The present study was designed to understand innate immune responses of farmed juvenile gilthead seabream (Sparus aurata) after Photobacterium damselae subsp. piscicida (Phdp) infection. Therefore, two groups of seabream juveniles were intraperitoneally injected with 100 µL of PBS (placebo) or 100 µL of exponentially growing Phdp (1 × 106 CFU/mL; infected). The blood, plasma, liver, and head kidney of six fish from each treatment were sampled immediately before infection and 3, 6, 9, 24 and 48 h after infection for the broad screening of fish immune and oxidative stress responses. Infected animals presented marked anaemia, neutrophilia and monocytosis, conditions that are correlated with an increased expression of genes related to inflammation and phagocytic activity. Similar studies with different fish species and bacteria can be useful for the definition of health biomarkers that might help fish farmers to prevent the occurrence of such diseases.

Activation of STAT and SMAD Signaling Induces Hepcidin Re-Expression as a Therapeutic Target for β-Thalassemia Patients.

Iron homeostasis is regulated by hepcidin, a hepatic hormone that controls dietary iron absorption and plasma iron concentration. Hepcidin binds to the only known iron export protein, ferroportin (FPN), which regulates its expression. The major factors that implicate hepcidin regulation include iron stores, hypoxia, inflammation, and erythropoiesis. When erythropoietic activity is suppressed, hepcidin expression is hampered, leading to deficiency, thus causing an iron overload in iron-loading anemia, such as β-thalassemia. Iron overload is the principal cause of mortality and morbidity in β-thalassemia patients with or without blood transfusion dependence. In the case of thalassemia major, the primary cause of iron overload is blood transfusion. In contrast, iron overload is attributed to hepcidin deficiency and hyperabsorption of dietary iron in non-transfusion thalassemia. Beta-thalassemia patients showed marked hepcidin suppression, anemia, iron overload, and ineffective erythropoiesis (IE). Recent molecular research has prompted the discovery of new diagnostic markers and therapeutic targets for several diseases, including β-thalassemia. In this review, signal transducers and activators of transcription (STAT) and SMAD (structurally similar to the small mothers against decapentaplegic in Drosophila) pathways and their effects on hepcidin expression have been discussed as a therapeutic target for β-thalassemia patients. Therefore, re-expression of hepcidin could be a therapeutic target in the management of thalassemia patients. Data from 65 relevant published experimental articles on hepcidin and β-thalassemia between January 2016 and May 2021 were retrieved by using PubMed and Google Scholar search engines. Published articles in any language other than English, review articles, books, or book chapters were excluded.

Notes on…

Abstract This month, Francesco Cian, of Batt Laboratories, presents a haematology case of a dog with marked anaemia.

Mcm2 Deficiency Leads to Bone Marrow Failure and Lymphoid Malignancies Dependent on Age and Genetic Background

DNA replicative stress is associated with malignant transformation. Mini-chromosome maintenance 2 (hereafter Mcm2) is a component of the DNA helicase complex that plays a pivotal role in DNA replication. The Mcm2-7 complex is loaded onto chromatin during the G1-phase of the cell cycle and is required for initiation of DNA replication in the subsequent S-phase.Mice with a Cre cassette inserted into the 3'UTR of the Mcm2 gene (designated Mcm2 Cre) were generated as a tool to assess Mcm2 expression. Unexpectedly, mice with two copies of this allele (Mcm2 Cre/Cre) had decreased expression of Mcm2 protein (~20-30% of the wild type (WT) expression level), likely due to decreased RNA stability caused by insertion of the Cre cassette. Surprisingly, all Mcm2 Cre/Cre mice developed a lethal pre-T lymphoblastic leukemia/lymphoma (pre-T LBL) with a median survival of under 3 months. These pre-T LBL were characterized by 10-20 interstitial deletions of approximately 50-1000 kb, often involving genes known to be mutated or deleted in human pre-T LBL, such as Notch1, Pten, Tcf3, and Cdkn2a. Thus, Mcm2 Cre/Cre mice display a novel mutator/deletor phenotype that results in malignant transformation.Although the Mcm2 hypomorph is a germline defect, the malignancies we identified in Mcm2 Cre/Cre mice were restricted to thymocytes. We hypothesized that Mcm2 Cre/Cre mice were susceptible to non-thymocyte malignancies, but due to the early onset of pre-T LBL (within 4 months of age), these putative non-thymocyte malignancies did not have an opportunity to develop. We transplanted Mcm2 Cre/Cre Lin-Sca-1+Kit+ (LSK) hematopoietic stem/progenitor cells (HSPCs) isolated from 5-week-old Mcm2 Cre/Cre bone marrow into WT recipients, which led to marked anemia and thrombocytopenia without evidence of leukemic transformation at 5 months post-transplant. Consistent with this finding, bone marrow from 5-week-old Mcm2 Cre/Cre mice also showed decreased LSK and Lin-Sca-1-Kit+ (LK) cells compared to WT mice, suggesting that the bone marrow failure was uncovered by the more prolonged survival of Mcm2 Cre/Cre LSK recipients vs. non-transplanted Mcm2 Cre/Cre mice (5 mos. vs. <3 mos.) .As an alternate approach to preventing pre-T LBL, we crossed the Mcm2 Cre allele onto a nude (nu/nu) background, as nude mice lack thymic epithelial cells, preventing T cell development. Mcm2 Cre/Cre nu/nu mice had markedly prolonged survival compared to Mcm2 Cre/Cre (median 296.5 vs. 80.5 days, respectively; P < 0.0001). Most of the Mcm2 Cre/Cre nu/nu mice succumbed to B-cell precursor acute lymphoblastic leukemia (BCP-ALL) within 10 months of life. To determine whether Mcm2 Cre/Cre nu/nu would develop acute myeloid leukemia (AML) if placed on myeloid leukemia sensitized background, we crossed Mcm2 Cre/Cre nu/nu mice with mice expressing a NUP98-PHF23 (NP23) transgene, as the NP23 fusion gene predisposes mice to develop AML. All Mcm2 Cre/Cre nu/nu NP23 mice died within 5 months of age, primarily due to BCP-ALL. Sparse whole-genome sequencing (WGS) was used to detect copy number aberration (CNA), and we identified recurrent deletions of 100-1000 kb that involved genes known or suspected to be involved in BCP-ALL, including Pax5, Ikzf3, Il7r, and Bcor. Whole-exome sequencing identified recurrent mutations, Jak1/Jak3, Ptpn11, and Kras.In an effort to identify 100-1000 kb interstitial deletions in non-hematopoietic tissue, we used sparse WGS to identify CNA in single-cell cloned mouse embryo fibroblasts (MEF) from Mcm2 Cre/Cre, Mcm2 Cre/Wt, and Mcm2 Wt/Wt mice. Very few CNAs were identified in any of the MEFs. We conclude that this unique deletor phenotype found in Mcm2 Cre/Cre mice is a powerful tool for the identification of tumor suppressor genes in lymphoid leukemia, and that B- and T- lymphocytes are uniquely susceptible to this deletor phenotype. DisclosuresNo relevant conflicts of interest to declare.

Bone Marrow Sinusoidal Endothelial Cells Are a Site of Fgf23 Upregulation in Iron Deficiency Anemia

Iron deficiency anemia (IDA) has been identified as a potent stimulator of FGF23 (fibroblast growth factor 23), a phosphaturic hormone classically thought to be produced by bone-embedded osteocytes. Recently, both phlebotomy and erythropoietin administration have been shown to upregulate FGF23 production in bone marrow. However, the cell type(s) mediating FGF23 upregulation in states of perturbed erythropoiesis require further clarification.Tmprss6-/- mice exhibit hepcidin elevation leading to systemic iron deficiency and iron-restricted anemia. We previously reported that Tmprss6-/- mice exhibit altered phosphate balance, elevated circulating FGF23, and Fgf23 mRNA upregulation in bone marrow but not cortical bone. Here, we clarify the sites of Fgf23 promoter activity in Tmprss6 -/- bone marrow using a reporter allele in which the enhanced green fluorescent protein (eGFP) coding sequence has been knocked into the endogenous Fgf23 locus. We generated Tmprss6 +/+,Tmprss6 +/-, and Tmprss6 -/- littermates of both sexes that carried either one (Fgf23 +/eGFP) or zero (Fgf23 +/+) copies of the reporter allele. Tmprss6-/- mice showed hyperhepcidinemia, hypoferremia, microcytic anemia, and tissue iron deficiency, which were not altered by heterozygous Fgf23 disruption (Figure 1A-C). By ELISA, Tmprss6-/- Fgf23 +/eGFP mice showed plasma levels of “total” FGF23 (intact, active hormone and C-terminal cleaved fragments) that remained markedly elevated compared to Tmprss6+/+ littermates (Figure 1D). Total FGF23 elevation in Tmprss6-/- Fgf23 +/eGFP mice was slightly less pronounced than Tmprss6-/- Fgf23 +/+ mice, suggesting an effect of Fgf23 gene dosage. In mice with 2 intact Fgf23 alleles, serum erythropoietin showed a strong linear correlation with plasma total FGF23.By confocal imaging, femurs of mice carrying the Fgf23 eGFP allele showed green fluorescence in vascular regions of the bone marrow but not in the bone cortex. Green fluorescence was more intense in Tmprss6-/- Fgf23+/eGFP mice than non-anemic controls. By flow cytometry of enzymatically digested bone marrow, we observed bright green fluorescence in a subset of endothelial cells (CD45 - Ter119 - CD31 +) exclusively in mice carrying the Fgf23 eGFP reporter allele (Figure 1E). The percentage of endothelial cells that were GFP bright was higher in Tmprss6-/- Fgf23 +/eGFP versus non-anemic mice. To clarify the endothelial cell subtype that expresses Fgf23, we mined published transcriptomic datasets from mice of normal iron balance and discovered higher Fgf23 mRNA in bone marrow sinusoidal endothelial cells compared to other bone marrow endothelial cell populations. Accordingly, we used anti-GFP immunohistochemistry in formalin-fixed bone marrow sections to assess Fgf23 eGFP reporter allele expression in the context of tissue architecture. Tmprss6-/- Fgf23 +/eGFP mice showed GFP expression in bone marrow sinusoidal endothelial cells, which was more intense than in non-anemic controls (Figure 1F). GFP reporter expression was also detected in rare cells of the thymus but not in liver, spleen, heart, muscle, or kidney. Collectively, our data reveal that bone marrow sinusoidal endothelial cells are a site of Fgf23 upregulation in chronic IDA.Because IDA in Tmprss6-/- mice results from pathologic hepcidin elevation, we also sought to determine if bone marrow sinusoidal endothelial cells are a site of Fgf23 upregulation in anemic mice with intact hepcidin regulation. We therefore subjected Fgf23 +/eGFP mice (with 2 intact Tmprss6 alleles) to a 500µl phlebotomy regimen (with saline volume replacement) known to induce marked anemia and hepcidin suppression. Compared to non-phlebotomized Fgf23 +/eGFP controls, phlebotomized Fgf23 +/eGFP mice showed severe anemia, elevated serum erythropoietin, and elevated plasma FGF23 18 hours after blood loss. Additionally, immunohistochemistry revealed more intense GFP expression in bone marrow sinusoidal endothelial cells of phlebotomized Fgf23 +/eGFP mice than non-phlebotomized controls. Taken together, our results show for the first time that bone marrow sinusoidal endothelial cells are a site of Fgf23 upregulation in both acute and chronic anemia. Given the serum erythropoietin elevation in both models, our findings suggest that erythropoietin may act directly or indirectly on sinusoidal endothelial cells to promote FGF23 production during anemia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.