Blood Marrow Research Articles

Cardioimmunology in Health and Diseases: Impairment of the Cardio-Spleno-Bone Marrow Axis Following Myocardial Infarction in Diabetes Mellitus

A comprehensive understanding of the cardio-spleen-bone marrow immune cell axis is essential for elucidating the alterations occurring during the pathogenesis of diabetes mellitus (DM). This study investigates the dynamics of immune cell kinetics in DM after myocardial infarction (MI) over time. MI was induced in diabetic and healthy control groups using C57BL/N6 mice, with sacrifices occurring at days 1, 3, 7, and 28 post-MI to collect heart, peripheral blood (PB), spleen, and bone marrow (BM) samples. Cell suspensions from each organ were isolated and analyzed via flow cytometry. Additionally, the endothelial progenitor cell-colony-forming assay (EPC-CFA) was performed using mononuclear cells derived from BM, PB, and the spleen. The results indicated that, despite normal production in BM and the spleen, CD45+ cells were lower in the PB of DM mice at days 1 to 3. Further analysis revealed a reduction in total and pro-inflammatory neutrophils (N1s) in PB at days 1 to 3 and in the spleen at days 3 to 7 in DM mice, suggesting that DM-induced alterations in splenic neutrophils fail to meet the demand in PB and ischemic tissues. Infiltrating macrophages (total, M1, M2) were reduced at day 3 in the DM-ischemic heart, with total and M1 (days 1–3) and M2 (days 3–7) macrophages being significantly decreased in DM-PB compared to controls, indicating impaired macrophage recruitment and polarization in DM. Myeloid dendritic cells (mDCs) in the heart were higher from days 1 to 7, which corresponded with the enhanced recruitment of CD8+ cells from days 1 to 28 in the DM-infarcted myocardium. Total CD4+ cells decreased in DM-PB at days 1 to 3, suggesting a delayed adaptive immune response to MI. B cells were reduced in PB at days 1 to 3, in myocardium at day 3, and in the spleen at day 7, indicating compromised mobilization from BM. EPC-CFA results showed a marked decrease in definitive EPC colonies in the spleen and BM from days 1 to 28 in DM mice compared to controls in vitro, highlighting that DM severely impairs EPC colony-forming activity by limiting the differentiation of EPCs from primitive to definitive forms. Taking together, this study underscores significant disruptions in the cardio-spleen-bone marrow immune cell axis following MI in DM, revealing delayed innate and adaptive immune responses along with impaired EPC differentiation.

Impact of Simultaneous Presence of Multiple PML-RARA Isoforms on Phenotype in Patients with Acute Promyelocytic Leukaemia.

To determine the coexistence of multiple PML-RARA transcripts in adult APL (acute promyelotic leukaemia) patients, and its impact on the patients' laboratory parameters, treatment responses, and prognoses. Cross-sectional study. Place and Duration of the Study: Department of Medical Genetics, Medical Faculty of Necmettin Erbakan University, Konya, Turkiye, from January 2015 to March 2023. The study group consisted of individuals diagnosed with APL. RNA isolation was performed by taking blood or bone marrow samples and the presence of breakpoints in PML-RARA bcr1, bcr2, and bcr3 was detected using the real-time PCR. However, the quantification of PML-RARA fusion transcripts cannot be provided using the utilised kit. Twelve women and eight men were examined with a mean age of 38 years (range: 19-80), and 46.5 years (range: 22-60) were examined, respectively. When evaluating patients based on isoforms, it was found that 40% had multiple isoforms. Nineteen (95%) patients achieved haematologic remission after the treatment. Only one patient who had three different isoforms did not achieve remission. The estimated median survival for patients with a single isoform and those with multiple isoforms was 78.1 months (95% CI: 37.8-117.6) and 71.7 months (46.2-97.2), respectively. Two of the patients with multiple isoforms were lost in the early stage, whereas no early-stage mortality was recorded among patients with a single isoform. Identifying PML-RARA isoform subtypes is important for predicting prognosis and informing clinical follow-up. Acute promyelocytic leukaemia, Breakpoint cluster region, Isoform, PML-RARA.

Establishment of human acute monocytic leukemia model with systemic infiltration in NPG mice.

A model construction of systemic acute leukemia is challenging. Herein, we established a systemic leukemia mouse model using highly immunodeficient NPG mice without any immunosuppressive treatments. NPG mice received tail intravenous injection of SHI-1 cells at the concentration of 1×107 cells (group A) or 5×107 cells (group B) and randomly sacrificed each seven days post-inoculation. Tumor development was monitored using nested-PCR, peripheral blood-smear analysis, flow cytometry, pathological examinations, and immunohistochemistry. The median survival of mice in groups A and B were 33.0 and 30.0 days, respectively. Blast cells in peripheral blood appeared on day 14 in group B, and on day 21 in group A. In addition, SHI-1 cell specific MLL-AF6 mRNA was detected in both spleen and bone marrow on day 14 post-inoculation. 21 days after inoculation, we observed human CD45+CD33+ cells with an SH-1-immunophenotype in the peripheral blood, spleen, and bone marrow, as well as solid neoplasms in multiple organs. Moreover, the histologically infiltrated leukemic cells expressed CD45. In conclusion, the current study demonstrated the normal growth of SHI-1 cells in the NPG mice without immunosuppression, which caused systemic leukemia similar to that observed in acute leukemia patients. We developed an efficient and reproducible model to study leukemia pathogenesis and progression.

How to diagnose acid sphingomyelinase deficiency (ASMD) and Niemann-Pick disease type C from bone marrow and peripheral blood smears.

How to diagnose acid sphingomyelinase deficiency (ASMD) and Niemann-Pick disease type C from bone marrow and peripheral blood smears.

Sterility of cord blood units: Evaluation of the collection, preparation and conservation process with a view to establishing a bank of stem cells from cord blood at the Biological Resources Center of the Institut Pasteur de Côte d’Ivoire (2024)

Introduction: Cord blood stem cells are used as an alternative to bone marrow and peripheral blood cell transplantation. For this purpose, cord blood banks have been established worldwide for the collection and cryopreservation of cord blood units. These banks must implement rigorous protocols to ensure the microbiological safety of grafts. The aim of this study is to evaluate the process of selecting mothers and collecting and processing these samples developed for this purpose. Methodology: Pregnant women were enrolled in the study on the basis of selection criteria. Maternal blood and cord blood samples were collected by the in utero method. Serological analyses of maternal blood for the detection of vertically transmitted infections and microbiological examinations of cord blood units were carried out. Results: 31 women out of 46 (68.4%) were enrolled. All had negative serology for the targeted pathogens. Similarly, microbiology revealed no microbial contamination of the cord blood units. Conclusion: This work demonstrates that the procedures for selecting mothers and processing implementations are effective in ensuring the microbiological safety of cord blood units and could be suggested in the context of banking cord blood stem cells at the Institut Pasteur de Côte d’Ivoire.

Hematological picture of pediatric Sudanese patients with visceral leishmaniasis and prediction of leishmania donovani parasite load.

Visceral leishmaniasis (VL) is a systemic protozoan infection caused by Leishmania donovani (L. donovani) and transmitted by sand flies, causing macrophage invasion in the liver, spleen, and bone marrow. Diagnosis of VL is currently based on clinical signs, symptoms, and specific in-vitro markers and bone marrow investigations. However, VL's specific hematological and bone marrow manifestation in Sudanese pediatric patients is not well studied. To examine the blood and bone marrow characteristics in pediatric patients from Sudan who have VL. This is a retrospective hospital-based study with a sample of 107 consecutive Sudanese pediatric patients. The data focused on hematological and bone marrow results. We included only the completed records of the pediatric patients with VL in the Tropical Disease Teaching Hospital in Khartoum, Sudan from the period of 2016 to 2020. The majority of pediatric patients included in this study are below 5-years-old (n = 59, 55.2%). Moreover, anemia, thrombocytopenia, and leukopenia were among the prevalent characteristics in the population under study. To further analyze the data, we developed a machine learning model using boosted forest algorithms to predict L. donovani parasites load, with a mean accuracy of 0.88 for the training dataset and an accuracy of 0.46, 0.50, and 0.74 for mild, moderate, and severe L. donovani parasite load in the validation dataset. This study shows that the most common bone marrow change among Sudanese VL children was increased chronic inflammatory cells (n = 88, 82.2%) with present macrophage hemophagocytes (n = 103, 96.3%). While anemia and thrombocytopenia were the most common hematological changes. These results will hopefully lead to an early diagnosis and hence better management for Sudanese pediatric patients with suspected VL.

Diagnosis of canine B-cell chronic lymphoid leukemia with a CD21 negative phenotype using the LT21 clone CD21 antibody in flow cytometry: a case report

BackgroundChronic lymphoid leukemia (CLL) is a hematological disorder characterized by the clonal expansion of small mature lymphocytes that accumulate in the blood and bone marrow. CLL can arise from B-, T-, or natural killer cell clones. The cytological evaluation of blood smears is often the simplest and least invasive method for diagnosing lymphoid leukemia. Immunophenotyping is used to further subclassify the type of lymphoid leukemia.Case presentationA 15-year-old, 4.4-kg spayed female Shih Tzu was presented to the veterinary medical teaching hospital of Kangwon National University. Despite having a normal appetite and activity level, cervical and inguinal lymph node enlargement was noted on physical examination. Complete blood count revealed severe leukocytosis, severe lymphocytosis, and monocytosis. Splenomegaly, hepatomegaly, and lymph node enlargement were detected on radiographic and ultrasonographic examination. Immunophenotyping was performed using peripheral blood mononuclear cells (PBMCs). The majority of lymphocytes exhibited the following profiles: CD3−CD79a− (97.5%), CD4−CD8− (98.6%), CD21−CD79a− (98.4%), CD34− (0.1%), CD45+ (99.6%), major histocompatibility complex class II+ (99.5%), and CD14− (0.5%). Based on the immunophenotyping results, possible differentials considered included the following: the majority of lymphocytes may be natural killer (NK) cell clones, plasma cell clones, or show aberrant expression or loss of CD21 marker due to the neoplastic nature of the cells. Further flow cytometry was performed using antibodies against CD3, CD5, CD94, and granzyme B. The combined results indicated that the predominant lymphocyte subset in the PBMCs was CD3−CD5−CD21−CD94−granzyme B−. To confirm monoclonality and exclude the aberrant loss of CD markers, a polymerase chain reaction for antigen receptor rearrangement (PARR) assay was conducted. The PARR assay, using DNA from blood and lymph node samples, showed B-cell monoclonality. Immunocytochemistry using PBMCs showed that the plasma cell marker Multiple Myeloma Oncogene 1 (MUM1) was not expressed. Therefore, the diagnosis was confirmed to be B-cell CLL.ConclusionImmunophenotyping can help subclassify the type of lymphoid leukemia; however, as tumor cells can show aberrant expression or loss of the CD21 marker, combining immunophenotyping with the PARR assay could yield a more accurate diagnosis.

Proteogenomic characterization of highly enriched viable leukemic blasts in acute myeloid leukemia: A SWOG report

AbstractIntroductionAcute myeloid leukemia (AML) remains one of the deadliest hematopoietic malignancies. A better understanding of the molecular biology governing AML may lead to improved risk stratification and facilitate the development of novel therapies. Proteins are responsible for much of the biology of cells. Several studies have examined the global proteome in bulk mononuclear cells (MNCs) from AML specimens, which are comprised a heterogenous population of cells at various stages of differentiation.MethodsGiven the potential impact of the nonleukemic cells on protein expression profiles, we applied an integrative proteogenomic approach utilizing next‐generation sequencing and mass spectrometry‐based proteomics to identify novel protein biomarkers in unsorted MNCs and viable leukemic blasts (VLBs) isolated from blood and bone marrow specimens obtained at the time of AML diagnosis.ResultsWe identified significant differences in protein expression between VLBs and MNCs. Subsequent studies (N = 27) focused on proteomic profiling of VLBs that identified novel candidate biomarkers associated with mutational genotypes and clinical outcome, some of which were recapitulated in an independent cohort of patients. Using mass spectrometry, we also detected mutated protein products, some of which were predicted via in silico analyses to be potential neoantigens amenable to adoptive immunotherapy. As previously described, analyses comparing transcript and protein expression showed an overall modest correlation between mRNA and protein dataset, but enriching for genes associated with mutations significantly improved the protein–RNA correlation.ConclusionTogether, the results provide insight into the biology of VLBs and demonstrate the gains derived from examining the proteome in addition to genome and transcriptome.

Future directions in myelodysplastic syndromes/neoplasms and acute myeloid leukaemia classification: from blast counts to biology.

Myelodysplastic syndromes/neoplasms (MDS) and acute myeloid leukaemia (AML) are neoplastic haematopoietic cell proliferations that are diagnosed and classified based on a combination of morphological, clinical and genetic features. Specifically, the percentage of myeloblasts in the blood and bone marrow is a key feature that has historically separated MDS from AML and, together with several other morphological parameters, defines distinct disease entities within MDS. Both MDS and AML have recurrent genetic abnormalities that are increasingly influencing their definitions and subclassification. For example, in 2022, two new MDS entities were recognised based on the presence of SF3B1 mutation or bi-allelic TP53 abnormalities. Genomic information is more objective and reproducible than morphological analyses, which are subject to interobserver variability and arbitrary numeric cut-offs. Nevertheless, the integration of genomic data with traditional morphological features in myeloid neoplasm classification has proved challenging by virtue of its sheer complexity; gene expression and methylation profiling also can provide information regarding disease pathogenesis, adding to the complexity. New machine-learning technologies have the potential to effectively integrate multiple diagnostic modalities and improve on historical classification systems. Going forward, the application of machine learning and advanced statistical methods to large patient cohorts can refine future classifications by advancing unbiased and robust previously unrecognised disease subgroups. Future classifications will probably incorporate these newer technologies and higher-level analyses that emphasise genomic disease entities over traditional morphologically defined entities, thus promoting more accurate diagnosis and patient risk stratification.

A plain language summary of the final analysis of the GRIFFIN study of daratumumab plus lenalidomide, bortezomib, and dexamethasone for people with newly diagnosed multiple myeloma.

This summary describes the final analysis of the GRIFFIN study. In this study, participants were newly diagnosed with a type of blood and bone marrow cancer called multiple myeloma, had never received any treatment, and were able to undergo an autologous stem cell transplant. The GRIFFIN study looked at adding the drug daratumumab (D) to a combination of standard treatments called RVd (lenalidomide [R], bortezomib [V], and dexamethasone [d]) during the treatment phases induction and consolidation, followed by daratumumab and lenalidomide (D-R) maintenance. Participants also received an autologous stem cell transplant to further help reduce multiple myeloma. The GRIFFIN study looked at whether D-RVd followed by D-R maintenance was better at killing multiple myeloma cells compared with RVd on its own followed by R maintenance on its own, and if treatments were safe. This summary also describes results from 2 other GRIFFIN publications: one that looked at participants with certain multiple myeloma characteristics or demographic factors that are associated with worse outcomes, and another that looked at how treatments impacted the participants' quality of life. At the time of the final analysis of GRIFFIN, participants who were treated with D-RVd followed by D-R maintenance had very low (undetectable) levels of multiple myeloma cells and multiple myeloma markers (biological signs) and were more likely to be alive without the multiple myeloma getting worse or coming back compared with participants who received standard RVd treatment followed by R maintenance. There was also a pattern of similar benefits achieved by participants who were at risk for worse outcomes. Additionally, participants who received D-RVd treatment followed by D-R maintenance reported less pain, less fatigue (extreme tiredness), and greater improvements in their ability to conduct daily physical activities. While some side effects (unwanted or unexpected effects of treatment) were higher with D-RVd, side effects in both groups were as expected, and adding daratumumab did not reduce a participant's ability to handle treatment. Results of the GRIFFIN study showed that D-RVd treatment followed by D-R maintenance was better at treating multiple myeloma than the standard treatment of RVd followed by R maintenance in adults with a new diagnosis of multiple myeloma who were able to receive an autologous stem cell transplant, with no unexpected side effects of treatment.Clinical Trial Registration: NCT02874742 (GRIFFIN) (ClinicalTrials.gov).

Diagnostic accuracy of bone marrow blood evaluation in haemophagocytic lymphohistiocytosis paediatric patients.

Haemophagocytic lymphohistiocytosis (HLH) is a rare and serious immunological syndrome that involves a strong activation of cytotoxic T lymphocytes and macrophages. HLH determines a cytokine-mediated tissue injury with a contemporary multi-organ failure and a high fatality rate. A retrospective study was performed considering the medical records of paediatric patients who underwent a bone marrow aspirate for suspect HLH. The biomarkers evaluated were among those included in the HLH-2004. Lactate dehydrogenase (LD) was also evaluated. Haemophagocytosis was evaluated in bone marrow blood smear slides. Enrolled were 11 patients included in the HLH group and 8 patients as controls. Haemoglobin and fibrinogen resulted lower in HLH patients than in controls, while blood triglycerides, serum ferritin and LD resulted increased. Blood triglycerides and fibrinogen discriminated HLH cases perfectly, with a sensitivity and specificity of 100%. Ferritin had a sensitivity of 100% and a specificity of 83% (cut off ≥3,721µg/L) and LD of 73% and of 100% (the cut off ≥1,903U/L). Haemoglobin was found to have a sensitivity of 75% and a specificity of 100% (cut off ≤ 96g/L). Total haemophagocytes cell counts were not different between patients and controls. Only the increased number of phagocytized nucleated red blood cells (NRBC) was found to be significantly increased in the patients. Erythrocytes phagocytosis (≥4/1,000 cells) only tended towards significance. The blood biomarkers showed better diagnostic performance than the morphological evaluation. Among the different cell lineages engulfed by haemophagocytes, the best diagnostic performance was obtained by phagocytosed mature erythrocytes and immature nucleated erythrocytes.

Antisalmonella Activities of Essential Oil of Psidium guajava Leaves: An In vitro and In vivo Study

Background: Life-threatening typhoidal salmonella infection is becoming more difficult to treat due to rising resistance to commonly used antibiotics. This resistance is aided by the biofilm-forming ability of the bacteria. This study investigated the in vitro and in vivo antisalmonella activities of essential oil of Psidium guajava leaves. Methods: Essential oil was extracted from the leaves of Psidium guajava with solvents. The sensitivity of Salmonella Typhi to the essential oil of Psidium guajava (EOPG) was assessed via the agar well diffusion method. Minimum inhibitory, bactericidal and biofilm-inhibitory concentrations (MIC, MBC and MBIC respectively) were measured using micro-broth dilution and crystal violet assay respectively. The effect of EOPG on the bacterial cell membrane was evaluated by measuring the efflux of potassium ions, inorganic phosphate and pyruvic acid. Bioautography and GC-MS were employed to identify the constituents of the active fraction of the EOPG. Effects of EOPG on the level of bacteremia and bone marrow infection in male Wistar rats were determined by plating caudal blood on salmonella-shigella agar. Selected serum cytokines were measured using ELISA kits. Effect on hepatic DNA was measured by comet assay while histological evaluation of the liver of infected rats was done by Hematoxylin and Eosin staining. Results: Essential oil of Psidium guajava leaves (EOPG) inhibited the growth of S. Typhi with a zone of inhibition of 13±0.2mm compared to 31±0.3mm recorded by ciprofloxacin. Minimum inhibitory, minimum bactericidal and minimum biofilm inhibitory concentrations were 25, >50 and 12.5 mg/mL respectively. Exposure to EOPG caused efflux of potassium ions, inorganic phosphate and pyruvic acid in S. Typhi. Oral administration of 50 mg/mL of EOPG reduced bacterial load in the blood and bone marrow of S. Typhi-infected rats significantly (P<0.05) when compared to untreated animals. Infection-induced reduction of serum IL-1β and rise in serum IFN-γ were ameliorated considerably in treated animals. The hepatic DNA of infected animals were protected from breakage in the group administered 50 mg/mL/day of EOPG compared with untreated, ciprofloxacin-treated as well as those administered 100 mg/mL/day of EOPG. Conclusion: EOPG has both in vitro and in vivo anti-salmonella activities, this could be exploited in developing new drug leads for treatment of typhoid fever

Along the gut-bone marrow signaling pathway: use of longan polysaccharides to regenerate blood cells after chemotherapy-induced myelosuppression.

Although it has been established that polysaccharides have an effect on bone marrow haematopoiesis, it remains unclear how polysaccharides regulate bone marrow haematopoiesis during absorption and metabolism in vivo. In this study, the effect of a longan polysaccharide of large molecular weight (TLPL) on the gut microbiota of mice and its implications for the haematopoietic process in bone marrow was discussed. Here, the results show that after 21 days of TLPL consumption, the respective quantities of white blood cells, platelets, hemoglobin and bone marrow nucleated cells were determined to be 3.18 ± 1.71 (109 L-1), 1238.10 ± 164.41 (109 L-1), 135.10 ± 4.95 (g L-1), and 1.70 × 107, which reached 56.98%, 117.28%, and 47.74%, respectively, of the results for NC. TLPL both increased the thymus and spleen indexes by up to 2.08 ± 0.64 (mg g-1) and 6.49 ± 2.45 (mg g-1), respectively. Additionally, TLPL remodeled the gut microbiota with a significant increase in Lactobacillus in particular, and a significant increase in the level of the potential intestinal metabolite lactate was detected in the serum. Most importantly, a similarly significant up-regulation of the gene expression of the lactate receptor, Gpr81, in the myeloid cells was observed. These changes contributed to the activation of the secretion of various cytokines associated with haematopoiesis, with the levels of G-CSF, EPO, SCF and PF4 increased by 2.44 times, 1.14 times, 1.56 times and 1.13 times, respectively, compared to the MC group, which subsequently accelerated production of bone marrow cells and blood cells. The findings of this study reveal the unique mechanism of dried longan polysaccharides in ameliorating myelosuppression and provide a feasible strategy for the treatment of chemotherapy-induced myelosuppression with bioactive polysaccharides.

PCR GeneScan Analysis of Rearranged Immunoglobulin or T-Cell Receptor Genes for Clonality Diagnostics in Suspect Lymphoproliferations.

Assessment of the presence of clonal lymphoproliferations via polymerase chain reaction (PCR)-based analysis of rearranged immunoglobulin (IG) or T-cell receptor (TR) genes is a valuable method in the diagnosis of suspect lymphoproliferative disorders. Additionally, this methodology can be used for evaluating dissemination of lymphoma cells and for studying the clonal relationship between multiple (different locations) and consecutive (over time) lymphomas. Here we describe an integrated approach to assess clonality via analysis of Ig heavy chain (IGH), Ig kappa (IGK), TCR beta (TRB), and TCR gamma (TRG) gene rearrangements, based on the standardized multiplex PCRs as originally developed by the European BIOMED-2 consortium (currently named EuroClonality). The described protocol covers the pre-analytical phase of DNA isolation (from formalin-fixed paraffin-embedded and fresh tissues, body fluids, peripheral blood, and bone marrow), the analytical phase of PCR GeneScan analysis, and the post-analytical interpretation of the obtained profiles, following established guidelines.

Osteopetrosis-like disorders induced by osteoblast-specific retinoic acid signaling inhibition in mice

Osteopetrosis is an inherited metabolic disease, characterized by increased bone density and narrow marrow cavity. Patients with severe osteopetrosis exhibit abnormal bone brittleness, anemia, and infection complications, which commonly cause death within the first decade of life. Pathologically, osteopetrosis impairs not only the skeletal system, but also the hemopoietic and immune systems during development, while the underlying osteoimmunological mechanisms remain unclear. Osteoclastic mutations are regarded as the major causes of osteopetrosis, while osteoclast non-autonomous theories have been proposed in recent years with unclear underlying mechanisms. Retinoic acid (RA), the metabolite of Vitamin A, is an essential requirement for skeletal and hematopoietic development, through the activation of retinoic acid signaling. RA can relieve osteopetrosis symptoms in some animal models, while its effect on bone health is still controversial and the underlying mechanisms remain unclear. In this study, we constructed an osteoblast-specific inhibitory retinoic acid signaling mouse model and surprisingly found it mimicked the symptoms of osteopetrosis found in clinical cases: dwarfism, increased imperfectly-formed trabecular bone deposition with a reduced marrow cavity, thin cortical bone with a brittle skeleton, and hematopoietic and immune dysfunction. Micro-CT, the three-point bending test, and histological analysis drew a landscape of poor bone quality. Single-cell RNA sequencing (scRNA-seq) of the femur and RNA-seq of osteoblasts uncovered an atlas of pathological skeletal metabolism dysfunction in the mutant mice showing that osteogenesis was impaired in a cell-autonomous manner and osteoclastogenesis was impaired via osteoblast-osteoclast crosstalk. Moreover, scRNA-seq of bone marrow and flow cytometry of peripheral blood, spleen, and bone marrow uncovered pathology in the hematopoietic and immune systems in the mutant mice, mimicking human osteopetrosis. Results showed that hematopoietic progenitors and B lymphocyte differentiation were affected and the osteoblast-dominated cell crosstalk was impaired, which may result from transcriptional impairment of the ligands Pdgfd and Sema4d. In summary, we uncovered previously unreported pathogenesis of osteopetrosis-like disorder in mice with skeletal, hematopoietic, and immune system dysfunction, which was induced by the inhibition of retinoic acid signaling in osteoblasts, and sheds new insights into a potential treatment for osteopetrosis.

Case Series: Unveiling mass-forming neoplastic extramedullary hematopoiesis in chronic myeloid leukemia

Abstract Introduction/Objective Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by BCR::ABL1 fusion and predominantly granulocytic proliferation. Three main phases of disease have previously been recognized: chronic, accelerated and blast phase, though accelerated phase has become controversial. During the chronic phase, leukemic cells exist predominantly in the peripheral blood and bone marrow; however, during blast phase, blasts can proliferate in the blood, bone marrow, or extramedullary tissues. Methods/Case Report We present two instances of cutaneous extramedullary hematopoiesis associated with CML at our institution. Results (if a Case Study enter NA) Case 1: A 34-year-old man previously diagnosed with CML presented with painful pustules on his right thigh. Biopsy revealed an infiltration of primarily left-shifted granulocytes, along with scattered megakaryocytes, and rare erythroid precursors. Immunohistochemical studies were consistent with maturing extramedullary hematopoiesis, devoid of sheets or clusters of blasts. Peripheral blood analysis revealed marked neutrophilic leukocytosis with basophilia and eosinophilia. Bone marrow examination depicted left-shifted granulocytic hyperplasia, megakaryocytic hyperplasia, and fewer than 5% blasts. Case 2: A 40-year-old man presented with fatigue, night sweats, weight loss, hepatosplenomegaly and palpable nodules on his left arm and thighs. The biopsy of the thigh nodule uncovered leukemic cells at different developmental stages, along with eosinophils, erythroid precursors, and megakaryocytes. Immunohistochemical staining confirmed their presence, with only a few blasts observed. Blood and bone marrow tests showed leukocytosis with blasts less than 5%, indicating CML in the chronic phase. Molecular analysis confirmed BCR-ABL1 fusion. Conclusion Mass-forming neoplastic extramedullary hematopoiesis in the skin during chronic phase of CML is a feature that is not well-documented in the literature. This entity shows immature granulocytic precursors, but does not exhibit an increase in blasts and therefore should not be called leukemia cutis or myeloid sarcoma. It is important to raise awareness of this entity to prevent misinterpretation as myeloid sarcoma and subsequent unnecessarily aggressive therapy.

Rare Adult acute leukemia with Coexisting myeloblastic (basophilic differentiation) & B-lymphoblastic subpopulations Case Study

Abstract Introduction/Objective Acute basophilic leukemia is a very rare subgroup of acute myeloid leukemia that is defined in the 2022 WHO classification by presence of at least 20% blast cells in peripheral blood or bone marrow with immature and maturing cells of basophil lineage. The simultaneous occurrence of two hematological malignancies in a middle- aged adult patient is quite a rare event as evidenced by the rare cases reported in the literature. To our knowledge, this is the first case to be reported with concomitant acute basophilic and B-lymphoblastic leukemia in adult patients. these findings necessitates the comprehensive systematic approach to attain accurate diagnosis and plan the appropriate treatment protocol. Methods/Case Report Bone marrow aspiration & biopsy procedure was performed under local anesthesia and marrow samples were subjected to: lieshman staining of marrow films and direct examination. Immunophenotypic analysis of marrow aspirate by FACS Canto II flow cytometer. Cytogenetics studies were done as karyotyping and gene mutation detection by FISH. Results (if a Case Study enter NA) NA Conclusion Morphology of marrow films: 50% Blast cells, group of them are medium to larg-cells with high nucleocytoplasmic ratio and immature chromatin and the other group shows blast cells with sparse basophilic granulations, plus 20% immatue and maturing basophils. IPT shows two populations: the Myeloblast population (basophilic differentiation) is Positive for CD45dim, CD34het, CD13, CD33, CD117prt, CD25het, CD123het, cyt MPO part, HLA-DR partial. the B lineage lymphoblast population is Positive for CD45dim, CD34het, CD19, CD79a, TdT, CD20het, CD38. Cytogenetics studies: hyperploidy 56, (X, Y, 9, 10, 12, 13, 15, 18, 22, mar+ extra-chromosomes) FISH: +ve for FLT3-ITD mutation. the picture is by morphology & IPT consistent with biclonal leukemia, myeloid clone (acute basophilic leukemia) & ProB-ALL. The systematic laboratory approach from careful morphological examination of peripheral blood and bone marrow smears to confirmatory immunophenotypic analysis of marrow specimens by flow cytometry, and the comprehensive cytogenetics analysis, has a vital role in diagnosing such rare and aggressive phenomenon and provide a good chance for planning the appropriate treatment protocol with critical timesaving and cost effectiveness.

Generation of Antibody Libraries for Phage Display: Human Fab Format.

Phage display is a powerful method for the de novo generation and affinity maturation of human monoclonal antibodies from naive, immune, and synthetic antibody repertoires. The pComb3 phagemid family of phage display vectors facilitates the selection of human monoclonal antibody libraries in the monovalent Fab format, which consists of human variable domains VH and VL (Vκ or Vλ), fused to the human constant domains CH1 of IgG1 and CL (Cκ or Cλ), respectively. Here, we describe the use of a pComb3 derivative, phagemid pC3C, for the generation of human Fab libraries with randomly combined human variable domains (VH, Vκ, and Vλ) of high sequence diversity, starting from the preparation of mononuclear cells from blood and bone marrow. Depending on the complexity of the parental antibody repertoire, the protocol can be scaled for yielding a library size of 108-1011 independent human Fab clones. As such, it can be used, for instance, for the generation of a large naive human Fab library from healthy individuals or for the generation of a specialized immune human Fab library from individuals with an endogenous antibody response of interest.

Myeloid sarcoma mimicking as recurrent squamous cell carcinoma: What is the etiology?

Abstract Introduction/Objective Myeloid sarcoma is an extramedullary malignancy of primitive myeloid cells and can occur during, preceding, or as a relapse of acute myeloid leukemia that occurs mostly in the soft tissue and skin. Methods/Case Report A 77-year-old white male with a history of oral squamous cell carcinoma status post resection (18 months ago) followed by radiation and chemotherapy (15 months prior), presented to the ear nose and throat clinic with increasing weakness and dizziness. He received two doses of Cisplatin and was currently on Pembrolizumab. On examination, there were two lesions in the oral cavity, one on the right gingivobuccal sulcus and a second on the left posterior mandibular ridge, suspicious for squamous cell carcinoma recurrence. Interestingly, the biopsy did not show evidence of recurrent squamous cell carcinoma but showed sheets of blasts that were positive for MPO, CD117, CD43, and CD68, consistent with myeloid sarcoma. Subsequent peripheral blood and bone marrow examinations showed acute myeloid leukemia. Results (if a Case Study enter NA) NA Conclusion Myeloid neoplasms post-cytotoxic therapy (MN-pCT) occur after radiation, alkylating agents, DNA topoisomerase II inhibitors, antimetabolites, and PARP1 inhibitors for a non-myeloid malignancy. Topoisomerase inhibitors are associated with MN-pCT after a latency period of 1 to 3 years; alkylating agents and radiation are associated with MN-pCT after a latency period of 5 to 7 years. Interestingly, this patient developed myeloid sarcoma in a previously irradiated area within 2 years. Cisplatin was discontinued after 2 doses due to side effect complications and is unlikely to be the culprit. Pembrolizumab is not considered to be a cytotoxic therapy and has not been reported to be associated with MN-pCT, however, the latency period for radiation-induced MN-pCT is unusually short in this patient and this raises the possibility of a Pembrolizumab-induced secondary myeloid neoplasm; additional investigation with other patients on this medication with second subsequent malignancies is required.

Severe Congenital Neutropenia with ELANE gene mutation, on G-CSF therapy; a case report

Abstract Introduction/Objective Severe congenital neutropenia (SCN) is a genetic syndrome characterized by a deficiency of mature neutrophils in the bone marrow and peripheral blood. The disease is caused by various gene mutations, with ELANE gene mutations accounting for 50% to 60% of cases. Methods/Case Report We report a case of a 12-year-old male patient with severe congenital neutropenia, positive for the ELANE gene mutation. He has a history of recurrent infections and unexplained fevers since childhood. He has been receiving G-CSF treatment for ten years but is now exhibiting a diminished response to it. Analysis of the bone marrow biopsy reveals trilineage hematopoiesis, maturing myeloid with a rise in eosinophils, maturing erythroid, megakaryocytes normal in number and morphology with significantly reduced iron reserves in normocellular bone marrow. On immunostaining, CD34, CD14, or CD117 stains were negative. Neither monocytes nor granulocytes were CD56 positive. Increased monocyte counts coincide with partial CD14 loss. Lymphocytes make up 10% of all cells and consist of Polytypic B-cells, natural killer cells, and a heterogeneous population of T-cells with a normal CD4 to CD8 ratio. Hence, myelodysplastic syndrome was ruled out. Results (if a Case Study enter NA) NA Conclusion This case report provides valuable insights into the limited knowledge of congenital neutropenia with ELANE mutation on G-CSF therapy. It highlights the importance of ruling out Myelodysplastic syndrome in patients with severe congenital neutropenia and ELANE gene mutation treated with G-CSF therapy.