Evaluation Of Bone Marrow Aspirates Research Articles

T Cell Ig and ITIM Domains (TIGIT) Expression in Multiple Myeloma

Background Neoplastic myeloma cells show a wide variety of mechanisms that induce the microenvironment, allowing immune evasion and promoting their proliferation, survival, migration and drug resistance. In this context, immunocheckpoints are particularly important, representing the modulators of signal pathways responsible for immune tolerance, a mechanism that prevents the destruction of cancer cells by the immune system. T cell Ig and ITIM domains (TIGIT), a member of the poliovirus (PVR) /nectin receptor family, is a new immune checkpoint that negatively regulates T cell functions. In Multiple Myeloma (MM), TIGIT could play a relevant role leading to immunological escape. TIGIT performs its task mainly through binding ligands: CD155 and CD112, through which it inhibits the cytotoxicity of CD8+ T cells, as well as that of NK. In This Study, we evaluate the presence of TIGIT in bone marrow using instant digital pathology. AIMS Aims of this study are to develop a valid and reproducible workflow for instant digital pathology, to demonstrate the presence of TIGIT-positive T lymphocytes in bone marrow of patients with MM and to evaluate its correlation with clinical features PATIENTS and Methods 18 patients with newly diagnosed MM not previously treated, were evaluated in this study. The sample was enrolled from November 2022 to May 2023. Bone marrow blood was loaded on a porous matrix able to retain cells (Cytomatrix, UCS Diagnostics srl, Rome) for real time assessment of sample adequacy, morphological details preservation, and composition of hematopoietic subpopulations, using an ex-vivo fluorescent confocal microscope (FCM). Expert pathologists compared the digital FCM images with permanent Formalin Fixed Paraffin Embedded (FFPE) sections of the matrix and trephine bone marrow biopsy. Immunohistochemistry for TIGIT (TG-1 Clone, OncoDianova) was performed on matrix FFPE sections. RESULTS Our results showed that 15/17 (88%) patients resulted positive for TIGIT and 2/17 (12%) were negative. TIGIT expression was found mainly on CD8+ T lymphocytes, TIGIT positivity was correlated with parameters that define the aggressiveness of myeloma disease such as PC > 60% or FLC > 100, ISS score, R-ISS score, cytogenetic risk and LDH > 220mU/ml. Among the patients defined as “ultra-High Risk”, 9/10 (90%) resulted positive for TIGIT. Comparing TIGIT with the MM International Staging System (ISS), we observed that all patients with scores II and III were positive for TIGIT (100%) while 78% of them with ISS I results positive. Conclusions Our results suggest that FCM can be applied immediate evaluation of bone marrow aspirates, preserving the cellular sample for final histological analysis. “Furthermore, our data shows the positivity of TIGIT in 88% of patients, demonstrating the presence of this immune checkpoint in the bone marrow microenvironment. These data will need to be confirmed in a larger group of patients, but they open up the possibility of new target treatments in multiple myeloma

A comparative study of bone marrow aspiration and trephine biopsy: Experience in a tertiary care center

Abstract Introduction: A bone marrow examination involves the pathological analysis of samples from the bone marrow, which are obtained through bone marrow aspiration (BMA) and biopsy procedures. This examination is employed for the diagnosis of various hematological and nonhematological conditions. Both aspirate and biopsy samples are essential, offering a comprehensive bone marrow assessment. Objectives: In this study, we aim to compare the simultaneous evaluation of bone marrow aspirates and biopsies and to study diagnostic accuracy and concordance rate. Materials and Methods: This research was performed at the Department of Pathology, Shri Aurobindo Medical College, and Postgraduate Institute in Indore, Madhya Pradesh, India. The study was conducted from January 2019 to December 2019 and evaluated 200 patients. In 170 instances, bone marrow biopsy (BMB) and BMA procedures were conducted simultaneously, with 14 cases excluded due to issues like hemodilution or inadequate samples. Microscopic examinations were carried out on both aspirate slides and trephine biopsy sections, with the findings meticulously documented. Results: In the present research, the most prevalent clinical reason for performing a bone marrow examination was anemia (28.2%), closely followed by acute leukemias (21.9%). Among the 156 cases examined, BMA and biopsy led to a definitive diagnosis in 116 patients (74.3%), while biopsy alone diagnosed 27 cases (17.3%). Conclusion: While BMB continues to be the established benchmark for bone marrow analysis due to its heightened reliability and sensitivity, the combined use of both BMA and BMB enhances diagnostic yield significantly.

Acute Erythroleukemia (AML-M6) in one-year-old boy: a case report

Background: Acute Erythroleukemia (AML-M6) is a rare case with approximately 3-4% of all Acute Myeloid Leukemia (AML) cases. AML-M6 is less common in the pediatric population, with an incidence of <1% of all pediatric leukemia cases. This case study aims to evaluate Acute Erythroleukemia (AML-M6) in a one-year-old boy at Prof. dr. IGNG Ngoerah Denpasar. Case Presentation: A one-year-old boy with clinical manifestations of fluctuating fever for a month, paleness, bleeding gums, bruising all over his body, swelling of both eyelids and splenomegaly. Peripheral Blood Smear (PBS) examination revealed bicytopenia with moderate normochromic normocytic anemia, severe thrombocytopenia and leukocytosis. A hypercellular picture was obtained from the examination of Bone Marrow Aspiration (BMA) with an increase in erythroblast activity of 74%, proerythroblast at 50% and normal myeloblasts activity at 10%. The patient was diagnosed with Acute Erythroleukemia (AML-M6). The patient died on day 16 of treatment. Conclusion: Based on the examination results, it was concluded that the patient was diagnosed with AML-M6 according to the PBS and BMA evaluation.

Diagnostic importance of bone marrow aspiration evaluation: A single-center study.

Background & Objective:Early diagnosis can be made based on the morphological examination of bone marrow aspiration (BMA) until the bone marrow biopsy (BMB) result is reported. This allows for treatment to be started immediately, especially in hematological malignancies for which urgent treatment is indicated. This study aimed to determine the sensitivity and importance of bone marrow aspiration in the diagnosis of hematological malignancies.Methods:In this study, the data of patients who underwent bone marrow aspiration and bone marrow biopsy in Van Yuzuncu Yil University hospital between 2017 and 2019 were retrospectively analyzed. A total of 500 patients who simultaneously underwent BMA and BMB were included in the study. Data were obtained from electronic medical records.Results:Indication for bone marrow evaluation was abnormalities in complete blood count in 270 (54%) of patients. The diagnosis was made based on the evaluation of BMA in 475 (95%). In 456 (96%) of the 475 patients diagnosed with BMA, the diagnosis was consistent with that of BMB. Agreement of BMB with BMA was 100% in acute and chronic leukemias, while BMA was not sufficient for the diagnosis of lymphoma and solid organ metastasis.Conclusion:Our study showed that the evaluation of BMA was highly sensitive in the diagnosis of hematological malignancies, such as acute leukemias, chronic leukemias, and multiple myeloma.

The Outcome Of Indonesian Protocol Chemotherapy Acute Lymphoblastic Leukemia 2018 Induction Phase at Dr. Soetomo Hospital

Background: The prognosis of pediatric acute lymphoblastic leukemia (ALL) has improved in the last 50 years. In Indonesia, national protocol of ALL chemotherapy is divided into 2 risk stratification, standard risk and high risk. The outcome after the induction phase of chemotherapy has an important role and has prognostic factor for the success of ALL chemotherapy. Objective: To determine the outcome of induction phase chemotherapy using the pediatric ALL 2018 national protocol at Dr. Soetomo Hospital Methods This prospective study was conducted from November 2020 to May 2021. Consecutive non-random sampling was done to children aged 1-18 years, recently diagnosed with ALL, and met the inclusion-exclusion criteria. Risk stratification was made based on age, initial leukocyte count, presence of a mediastinal mass, presence of CNS leukemia, and immunophenotyping. Evaluation of bone marrow aspiration was done after the sample had completed the induction phase of ALL chemotherapy according to the 2018 ALL national protocol stratification. Results: Twenty-one children recently diagnosed with ALL and underwent chemotherapy. Fourteen children underwent standard risk national protocol of ALL chemotherapy, and 7 remaining with high risk. Fifteen children completed the induction phase of chemotherapy, 6 remaining children died before completing. All patient underwent standard risk chemotherapy can complete induction phase. All patients who completed the induction phase showed remission on bone marrow aspiration evaluation. Conclusion: Patients who underwent standard risk of Indonesian protocol chemotherapy ALL 2018 have better outcome than high risk.

Retinal arterial macroaneurysm in leukemia

Retinal arterial macroaneurysm (RAM) is an acquired vascular dilatation usually seen at the site of arterial bifurcation or arterial-vein crossing. It's often seen in the first three branches of the central retinal artery. A 15-year-old male patient was admitted to the outpatient clinic with complaints of weakness, headache, and bruising on the body for a few weeks. During the physical examination, a hepatomegaly and a common patchy ecchymosis in the body were detected at the abdominal examination. Hemoglobin was 6.6 g/dl, white blood cells were 40,000/mm3, platelet was 24,000/mm3 at the whole blood count done. The 70% blast was detected in peripheral spread. Bone marrow aspiration evaluation was consistent with pre-B cell acute lymphoblastic leukemia. There was two macroaneurysms in the inferior arcuate of the left eye. The patient was examined on a weekly basis. During control examinations, retinal macroaneurysms in left eye and the hemorrhages significantly regressed. The fact that RAM is not often seen in the pediatric age group, indicates the importance of this phenomenon. It should be remembered that a complete ophthalmic examination should be performed on leukemia patients.

Spectrum of myelodysplastic syndrome in patients evaluated for cytopenia(s). A Report from a Reference Centre in Saudi Arabia

Background/ObjectiveMyelodysplastic syndrome (MDS) is a clonal disorder of hematopoietic stem cells, characterized by ineffective hematopoiesis, peripheral cytopenias along with hypercellularity of the bone marrow, and marked dysplastic features. Establishing MDS diagnosis is difficult due to nonspecific clinical presentation and imprecise morphological criteria. In anticipation to improve the diagnostic approach in this field, we aimed to characterize the clinical and morphological features of patients presented with cytopenias with a special focus on MDS. MethodsWe comprehensively reviewed all medical record of patients who were referred to the hematology laboratory at KFSH-RC, Riyadh, Saudi Arabia, between January 2009 and March 2016 for evaluation of bone marrow aspirates and trephine biopsies due to severe and persistent cytopenia(s) to rule out MDS. ResultsA total of 183 patients, 155 adult and 28 pediatric, were identified. In the adult group, MDS was diagnosed in 82 (52.9%) patients, with a male-to-female (M:F) ratio of 1.6:1 and mean age at diagnosis of 50 years. According to the World Health Organization (WHO) 2017 criteria, MDS subtypes were as follows: MDS with single lineage dysplasia (SLD, 5%), MDS with ring sideroblasts and SLD (MDS-RS-SLD 7%), MDS with multilineage dysplasia (MDS-MLD 21%), MDS with deletion of chromosome 5q (MDS del(5q), 2%), MDS unclassifiable (MDS-U7%), hypoplastic MDS (h-MDS 4%), MDS with excess blasts-1 (MDS-EB1, 20%), MDS with excess blasts-2 (MDS- EB2, 28%), and therapy-related MDS (6%). Laboratory and morphological features were described. In both groups, cytogenetic abnormalities were classified according to the Revised International Prognostic Scoring System cytogenetic risk groups. In adults, the dominating cytogenetic abnormalities were monosomy 5 and monosomy 7 seen in 20.7% and 24.4% of patients, respectively. Peripheral cytopenia not due to MDS was diagnosed in 54 (34.8%) patients, with a mean age of 43 years and M:F ratio of 1:1. The cause of these cytopenias were as follows: bone marrow failure (BMF, 22%), peripheral destruction (20%), drug induced (20%), anemia of chronic disease (16%), B12 deficiency (7%), infection (7%), paroxysmal nocturnal hemoglobinuria (4%), idiopathic cytopenia of undetermined significance (2%), and idiopathic dysplasia of undetermined significance (2%). A definite diagnosis of MDS was not possible in 19 patients due to insufficient clinical data. In the pediatric group, MDS was diagnosed in 14/28 (50%) patients, with M:F ratio of 1.8:1 and mean age at diagnosis of 4 years. MDS subtypes (WHO 2017) in 14 patients were as follows: refractory cytopenia of childhood (RCC, 42.8%), MDS-EB1 (42.8%), and MDS-EB2 (14.2%). Laboratory and morphological features were described. The prevalent cytogenetic abnormality was monosomy 7 in six/14 (42.8%) patients. Cytopenias due to other causes were diagnosed in eight/28 patients (28.5%), with a mean age of 6.5 years and M:F ratio of 1.6:1. The causes of non-MDS related cytopenia were: congenital BMF (4 patients), peripheral destruction (2 patients), immune deficiency (1 patient), and viral infection (1 patient). A definite diagnosis of MDS could not be made in six/28 (21.4%) patients. ConclusionMDS is the cause of cytopenia in a significant number of patients referred for evaluation of cytopenias, appears at younger age, and tends to be more aggressive than that reported in international studies. Anemia, dysplastic neutrophils in the peripheral blood, and dysplastic megakaryocytes in the bone marrow trephine biopsy are the most reliable features in distinguishing MDS from other alternative diagnoses.

AML: Negative Prognostic Impact of Early Blast Persistence Can be Overcome By Subsequent Remission Induction

Introduction: In acute myeloid leukemia [AML] intensive treatment usually includes an interim bone marrow assessment in the context of the first induction chemotherapy cycle. Early blast clearance has been shown to predict for achievement of a complete remission [CR], which makes the interim bone marrow result a widely used additive for further therapy decision making. However, there is an ongoing debate on the prognostic role of early blast clearance (day 14 - 21) and consistent clarification of this issue is still lacking. Here, we provide monocentric data from a large intensively treated AML cohort addressing the prognostic impact of early remission status on long-term survival. Methods: Data of 1008 intensively treated AML patients who were diagnosed between 2000 and 2017 and aged between 18 and 86 years were retrieved from our local AML database. Evaluation of bone marrow aspirates and / or biopsies was conducted by cytologic, flow cytometric and histopathological methods. Remission status was determined in accordance with the European-Leukemia Net [ELN] criteria of 2017. Patients were grouped according to their remission status after induction 1 (day 14 - 21) and prior to further therapy (chemotherapy and / or allogeneic hematopoietic stem cell transplantation [HSCT]). Baseline characteristics of the two groups such as age, cytogenetic / molecular risk according to ELN 2010, performance score according to Eastern Cooperative Oncology Group [ECOG] and Charlson Comorbidity Index [CCI] were compared using chi-square test and Kruskal-Wallis H test followed by post-hoc testing. Overall- and relapse-free-survival [OS, RFS] were analyzed using Kaplan-Meier- and Cox regression models. Results: In 57 % (n = 572) of the entire cohort, complete / incomplete remission [CR / CRi] or at least morphologic leukemia free state [MLFS] were observed after the first cycle of induction therapy. Partial response [PR] was found in 19 % (n = 196) and persistent disease [PD] in 24 % (n = 240) of all patients. The three different groups (CR / CRi / MLFS vs. PR vs. PD) did not significantly differ in age, ECOG-score or CCI, but - as expected - substantially with regard to ELN risk classification. Of the entire cohort, 62 % (n = 626) received a double induction, 61 % (n = 617) consolidation chemotherapy and 47 % (n = 477) allogeneic HSCT as a part of first-line therapy. Prior to HSCT, 85 % (n = 406) of the patients were in first CR / CRi / MLFS. Amongst these 406 patients, 51 % (n = 243) had only achieved PR / PD in early bone marrow puncture on day 14 - 21 of induction therapy. 71 % of these patients (n = 173) were converted into CR / CRi / MLFS prior to HSCT by additional chemotherapy. Within the non-transplanted group, 58 of 130 patients (44 % with initial PR / PD) achieved CR / CRi / MLFS after an additional cycle of induction chemotherapy. Early blast persistence and refractory disease were generally associated with inferior OS as compared with blast clearance (p < 0.001). Interestingly, early PR lost its negative prognostic impact on survival after conversion into CR / CRi / MLFS by additional therapy prior to post-induction therapy. This could particularly be observed in patients undergoing HSCT, but also in non-transplanted patients receiving a second cycle of induction therapy. In patients without allogeneic HSCT, early PR with blast clearance beyond induction 1 resulted in impaired RFS (p = 0.045) but not OS, possibly due to the influence of subsequent salvage therapy. Early PD remained prognostically unfavorable for OS and RFS in both transplanted and non-transplanted patients even after subsequent conversion into CR / CRi / MLFS (p = 0.002 and p = 0.021). Conclusion: Our results show that the achievement of CR / CRi / MLFS prior to post-remission therapy overrides the negative impact of an early partial response on OS, irrespective of the favorable prognostic impact that early blast clearance generally has on OS. With regard to OS, our results suggest a predominant role of the final remission quality after induction therapy. However, the adverse impact of achieving no response at interim bone marrow assessment can apparently not be attenuated by a later remission induction. On the assumption that the final depth of remission after induction therapy is predictive for OS, MRD-guided decision making prior to consolidation treatment might further improve first-line AML therapy, particularly in the intermediate ELN risk group. Disclosures Bullinger: Abbvie: Honoraria; Pfizer: Honoraria; Novartis: Honoraria; Menarini: Honoraria; Janssen: Honoraria; Hexal: Honoraria; Gilead: Honoraria; Daiichi Sankyo: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Astellas: Honoraria; Amgen: Honoraria; Bayer: Other: Financing of scientific research; Seattle Genetics: Honoraria; Sanofi: Honoraria; Jazz Pharmaceuticals: Honoraria.

14. A novel cytogenomic approach to diagnosis of plasma cell neoplasms

Plasma cell neoplasm (PCN) is characterized by specific chromosomal aberrations with diagnostic and prognostic significance. Low percentage of neoplastic cells is a drawback in application of karyotyping and fluorescence in situ hybridization (FISH) for evaluation of bone marrow aspirates. Enrichment techniques to isolate the CD138+ cells have improved the detection rates. However, a complete FISH analysis for various abnormalities may not be achieved due to limited number of isolated cells. In 2017, we validated and implemented a novel approach for a whole genome profiling of plasma cells in PCN. The protocol consisted of isolation of CD138+ cells, whole genome DNA amplification and microarray comparative genomic hybridization. Separate FISH analysis was performed for detection of balanced IGH rearrangements. Of 412 clinical cases to date, 234 (56.8%) had morphologic and immunohistochemical studies indicative of PCN with 0.5–90% plasma cells in a bone marrow aspirates. Among PCN patients, chromosomal abnormalities were identified in 223 (95.2%) demonstrated by both microarray and FISH (n = 76, 32.9%), microarray (n = 116, 50.2%) or FISH only (n = 31, 13.9%). The most common findings included IGH rearrangement (49%), 13q deletion (45%), hyperdiploidy (41%), and 1q duplication (38%). Tetraploidy and MYC rearrangements were observed in 2.1% (5/234) and 1.7% (4/234) of patients, respectively. Interestingly, microarray revealed characteristic deletions involving the 6q21q25 region in 4/7 patients with Waldenström Macroglobulinemia. Our diagnostic approach to PCN is a cost-efficient replacement of multiple FISH assays that provides a superior detection rate and additional information for disease classification, prognosis, treatment and differentiation from other B cell malignancies.

Clinical, haematological and bone marrow aspiration evaluation in Megaloblastic anemia - study of 80 cases in a tertiary care hospital Telangana

Clinical, haematological and bone marrow aspiration evaluation in Megaloblastic anemia - study of 80 cases in a tertiary care hospital Telangana

Bone Marrow Aspiration Evaluation in Clinical Management of Anemia Among Low Socioeconomic Group In A Tertiary Care Hospital.

Bone Marrow Aspiration Evaluation in Clinical Management of Anemia Among Low Socioeconomic Group In A Tertiary Care Hospital.

Evaluation of bone marrow aspirates in patients with acute myeloid leukemia at day 14 of induction therapy.

BackgroundEarly assessment of response to chemotherapy in acute myeloid leukemia may be performed by examining bone marrow aspirate (BMA) or biopsy (BMB); a hypocellular bone marrow sample indicates adequate anti-leukemic activity. We sought to evaluate the quantitative and qualitative assessment of BMA performed on day 14 (D14) of chemotherapy, to verify the inter-observer agreement, to compare the results of BMA and BMB, and to evaluate the impact of D14 blast clearance on the overall survival (OS).MethodsA total of 107 patients who received standard induction chemotherapy and had bone marrow samples were included. BMA evaluation was performed by two observers using two methods: quantitative assessment and a qualitative (Likert) scale. ROC curves were obtained correlating the BMA quantification of blasts and the qualitative scale, by both observers, with BMB result as gold-standard.ResultsThere was a significant agreement between the two observers in both the qualitative and quantitative assessments (Kw = 0.737, p < 0.001, and rs = 0.798, p < 0.001; ICC = 0.836, p < 0.001, respectively). The areas under the curve (AUC) were 0.924 and 0.946 for observer 1 and 0.867 and 0.870 for observer 2 for assessments of the percentage of blasts and qualitative scale, respectively. The best cutoff for blast percentage in BMA was 6 % and 7 % for observers 1 and 2, respectively. A similar analysis for the qualitative scale showed the best cutoff as “probably infiltrated”. Patients who attained higher grades of cytoreduction on D14 had better OS.ConclusionsEvaluation of D14 BMA using both methods had a significant agreement with BMB and between observers, identifying a population of patients with poor outcome.

Impact of Donor and Recipient Sex on Outcomes of HLA-Identical Sibling Allogeneic Hematopoietic Stem Cell Transplantation

Background: hematopoietic cell transplantation (HCT) is well established as therapy for hematologic malignancies as well as many non-malignant disorders. Donor selection is an important way to decrease the risks after HSCS and is therefore a key component of the clinical practice of transplantation. There are many criteria proved or hypothesized to affect outcomes after SCT and one of these, sex is the most controversial. Some investigators have found an increased risk of acute or chronic graft-versus-host disease (GVHD) associated with donor sex. Transplantation of stem cells from a female donor to a male recipient is a special circumstance in which donor T cells specific for minor H antigens, encoded by genes on the recipient Y-chromosome that are polymorphic to their X-chromosome homologues, may make a contribution to GVHD and GVL activity.Objective: investigate the effect of donor and recipient sex mismatch on outcomes of HLA-identical sibling allogeneic stem cell transplantation.Methods: this study was carried on 82 patients selected from patients who had underwent a myelo-ablative preparative regimen for a hematologic malignancy and received HSC transplant from an HLA-identical sibling being divided into 2 groups: 1st one with a female donor to a male recipient and 2nd one include otherwise. Both groups will be subjected to HLA tissue typing, CBC, Bone marrow aspirate evaluation, PT & PTT, blood culture when needed, SGPT, SGOT, ALP, blood urea, s.creatinine, urine analysis, pulmonary function tests, chest x-ray, echocardiography ECG, CMV-, EBV-, VZV-, Toxoplasma- antibodies (IgG , IgM), HBs-Ag, HBs-Ab, and HCV-Ab.Results: after variables controlling for patient and donor, there was an increased risk of acute GVHD in patients who received grafts from female donor being 18% for patients receiving grafts from female compared with 8% for recipients of male grafts. Donor sex was also an important influence on risk of developing chronic GVHD with cumulative incidences of chronic GVHD 9.7% for patients receiving transplants from male donors grafts compared to 24.3% for recipients of female donor grafts.Conclusion: we found that male recipients of grafts from female donors had a significantly higher probability for acute and chronic GVHD than male recipients with male donors or female recipients with female donors and despite the beneficial GVL effect observed in male recipients with female donors, these patients had significantly reduced survival compared with all patient/donor sex combinations, suggesting that other variables, particularly GVHD, contribute to transplantation-related mortality.

Diagnosis and treatment of chronic lymphocytic leukemia in a bat-eared fox (Otocyon megalotis)

Severe lymphocytosis and leukocytosis were detected during examination of a 10-year-old sexually intact male bat-eared fox (Otocyon megalotis) with regionally extensive alopecia. A CBC revealed severe leukocytosis (39,100 leukocytes/μL) and marked lymphocytosis (90%). A blood smear consisted predominantly of intermediate-sized lymphocytes and few large lymphocytes, with mild to moderate nuclear atypia. These findings were highly suggestive of chronic lymphocytic leukemia (CLL). Cytologic evaluation of bone marrow aspirates revealed no evidence of overt malignancy, with 10% of all cells identified as small to intermediate-sized mature lymphocytes. Treatment with chlorambucil and prednisone administered orally over a 1.8-year period decreased the leukocyte and lymphocyte counts to within reference intervals with no adverse effects. Although repeated flow cytometry revealed evidence of residual disease, the fox remained free of clinical disease, and WBC counts were within reference intervals for this species. At 22 months after initial evaluation, the fox was euthanized because of debilitating arthritis. No evidence of CLL was detected grossly or histologically during necropsy. To the authors' knowledge, this was the first report of CLL in a bat-eared fox and first successful treatment in a nondomestic carnivore. Treatment in accordance with a chemotherapeutic protocol successfully resolved the leukocytosis and lymphocytosis with no serious adverse effects. Description of this fox and the treatment protocol should provide a valuable reference for future cases in this and other nondomestic canine species.

Erythrocyte dysplasia in peripheral blood smears from 5 thrombocytopenic dogs treated with vincristine sulfate

Secondary dyserythropoiesis has been associated with vincristine administration in dogs. Evaluation of bone marrow aspirates for the presence of morphologic abnormalities in the erythroid lineage aids in the diagnosis. However, morphologic features of circulating erythroid precursors in these cases have not been described previously. The purpose of this report was to describe the cytologic features of dyserythropoiesis in peripheral blood and also bone marrow smears in a case series of dogs with immune-mediated thrombocytopenia (IMT) treated with vincristine sulfate. Nineteen dogs receiving vincristine for treatment of IMT were identified by retrospectively searching a computerized medical record system. There were 5 dogs that had dysplastic erythroid precursors in peripheral blood smears within 7 days of vincristine treatment. Two of those 5 dogs also had evidence for erythrodysplasia in modified Wright's-stained bone marrow smears obtained postvincristine administration. Morphologic changes included bizarre or inappropriate mitotic figures, abnormal nuclear configurations (fragmentation, elongation, indentation, and binucleation), atypical nuclear remnants (Howell-Jolly bodies), or nuclear and cytoplasmic asynchrony within the erythroid precursors. A brief review of the literature with discussion of the etiologies for dyserythropoiesis is provided. The dyserythropoiesis was clinically insignificant in all 5 cases and resolved. However, pathologists and clinicians should be aware of these potential findings to prevent misdiagnosis of other conditions.

Evaluation of bone marrow aspirates from multiple sites for staging of canine lymphoma and mast cell tumours

This prospective study evaluated the utility of bone marrow aspirates (BMAs) obtained from multiple sites for staging of canine lymphoma (LSA) and mast cell tumours (MCTs). Forty dogs (LSA, n = 24; MCTs, n = 16) were enrolled, but only 33 (82.5%) had diagnostic bone marrow (BM) aspirates obtained from two sites for inclusion in the study. Nineteen dogs with LSA were included, and 6 (31.6%) had BM involvement. Neoplastic lymphocytes were present in BM from both sites in all of these dogs. Fourteen dogs with MCTs were included, and 3 (21.4%) had BM involvement. Neoplastic mast cells were present at both sites in two dogs and at only one site in the third. These results indicate that BMAs from multiple sites may not be needed for accurate staging of canine LSA patients, but more studies evaluating the pattern of BM infiltration in dogs with high-grade MCTs are warranted.

CD81: A Novel, Specific and Highly Sensitive Marker in Flow Cytometric Diagnosis of Plasma Cell Dyscrasia

Abstract 2880 Background:The percent abnormal plasma cells (aPC) as determined by flow cytometry (FC) has been shown to be an independent risk factor for progression from myeloma precursor disease (monoclonal gammopathy of uncertain significance, MGUS; smoldering multiple myeloma, SMM) to multiple myeloma (MM). However, differentiation of aPCs from normal PCs (nPCs) in these patients is challenging. MM cell lines are know to underexpress the tetraspanin proteins (e.g. CD81, CD82) in comparison to nPCs. Although CD81, a nonglycosylated tetraspanin, is robustly expressed on the surface of nPCs, little information is available regarding its expression in the aPCs of MM, SMM and MGUS. In this study we evaluate the expression of CD81 in conjunction with CD19, CD45 and CD56 in bone marrow aPCs and nPCs from patients with MM, SMM and MGUS. Methods:Bone marrow aspirates from 41 patients (9 MGUS, 22 SMM, 7 MM, 3 non-neoplastic with clinical suspicion of MGUS) were analyzed with 8-color multiparametric FC using a panel of antibodies (CD138, CD38, CD19, CD20, CD27, CD28, CD45, CD56, CD81, CD13, CD14, CD16, CD3, CD34 and intracellular kappa & lambda light chains). The pattern of surface antigen and intracellular light chain expression was utilized to determine the percent aPC (defined as monoclonal with aberrant antigen expression) and percent nPC (defined as polyclonal with normal antigen expression). In all cases the pattern of antigen expression was evaluated in the aPCs; additionally, in cases with greater than 5% nPCs (19/41 patients: 8 MGUS, 8 SMM and 3 non-neoplastic) the pattern of antigen expression was evaluated in the nPCs. The ability to detect clonal aPC by evaluation of FC pattern of antigen expression was determined and compared for CD19, CD45, CD56 and CD81. We also examined the sensitivity and specificity of the CD19 and CD81 combination verses the conventional combination of CD19, CD56 and CD45 (Perez-Persona et al, Blood 2007) for the detection of clonal aPC. Results:CD81 was strongly expressed by nPC (average mean fluorescent intensity (MFI): 11500, standard deviation (SD): 5061, range: 5347–21657) in contrast to aPC with abnormally weak expression (average MFI: 1487, SD: 887, range: 647–4311). CD81 was a highly reliable marker for the detection of clonal PC; with 90% sensitivity and 100% specificity. It was the most specific and second most sensitive marker in our study (Table 1). CD81 was equally sensitive in detection of aPCs in MGUS, SMM and MM. Evaluation of the combined pattern of expression of CD19 and CD81 resulted in 100% sensitivity and 100% specificity for detection of aPC, which is greater than the conventional combination of CD19, CD56 and CD45, yielding 100% sensitivity but 90% specificity, for diagnostic evaluation of aPC. Conclusions:CD81 is a highly reliable marker in the detection of abnormal plasma cells in MM, SMM and MGUS. The combined approach of CD19 and CD81 is superior to other conventional marker combinations (i.e. CD19, CD45, and CD56) in terms of detection of clonal plasma cells and may replace their use in the clinical evaluation of bone marrow aspirates for plasma cell processes. Furthermore, it should help widening the applicability of minimal residual disease testing in MM.Table 1Sensitivity and specificity of different flow cytometric markers in the detection of malignant plasma cells.Sr NoMarkerSensitivitySpecificity1CD8190%100%2CD19100%84%3CD5668%90%4CD4576%53%5CD2779%95%6CD2824%100%7CD2024%90% Disclosures:No relevant conflicts of interest to declare.

Terminal Differentiation of FLT3/ITD AML Blasts In Patients Treated with the FLT3 Inhibitor AC220

AbstractAbstract 660AML is characterized by abnormal proliferation of myeloid cells that have a block in differentiation. FLT3/ITD mutations are relatively common in AML, and previous in vitro studies have demonstrated that signaling from ITD-mutated FLT3 blocks myeloid differentiation through repression of CEBP/a. As part of an ongoing phase 2 trial, we treated 6 patients with FLT3/ITD AML who were refractory to either primary induction therapy or salvage therapy after relapse with the highly potent and selective FLT3 inhibitor AC220. At the start of therapy, all 6 patients had circulating blasts (mean 9864 blasts/mm3; median 2970) and the median blast percentage in the bone marrow was 71.5%. Western blotting revealed a high level of sustained in vivo FLT3 inhibition in all patients. By Day 8, no patient had detectable blasts in the peripheral blood. After 14 days of treatment with AC220, all 6 patients displayed striking differentiation to the myelocyte stage within the bone marrow. By light microscopic evaluation of bone marrow aspirates, myelocytes (promyelocytes, myelocytes, and metamyelocytes) increased from a median of 10.5% pre-treatment to 52% after 2 weeks. Most patients were neutropenic on Day 1 of treatment (mean 574, median 560 neutrophils/mm3), but rose to a mean of 3275 neutrophils/mm3 after 4–8 weeks of treatment (median time to peak 34 days). By Day 28 of treatment, marrows were most often still hypercellular, but consisted primarily of fully differentiated neutrophils. Marrow blasts were markedly reduced or absent by Day 28 in all 6 cases (mean 2.3%, median 1.5%). In all 6 patients the FLT3/ITD mutation originally detected at the beginning of treatment was present in the marrow and peripheral blood despite the absence of circulating blasts after the first week of therapy. The FLT3 mutant allelic ratio did not change between pre-therapy and Day 28. Neutrophils were isolated to homogeneity (confirmed by cytospin) from peripheral blood by double ficoll density centrifugation. Using genomic DNA obtained from these purified neutrophils, we confirmed by PCR that the FLT3/ITD mutation was present, at a similar ratio as compared with the pre-treatment blasts. However, there was no detectable expression of FLT3 either by RNA (quantitative PCR) or protein (western blotting and flow cytometry) in these neutrophils. The isolated neutrophils morphologically resemble normal neutrophils by light microscopy, and by flow cytometry they express the differentiation antigen CD15 and CD11b, and have lost expression of immature markers such as cKIT and CD34. Stimulation of these neutrophils by endotoxin results in normal respiratory burst activity, as measured by reduction of nitroblue tetrazolium. They also express lactoferrin and MMP-9, proteins typically expressed in mature neutrophils. Clinically, lung nodules and fever occurred in 3 of the 6 patients within 14 days of the peak neutrophil count. They were not treated with steroids, but rather with antibiotics, and in all cases resolved. Other patients on this trial have developed Sweet's syndrome during the neutrophil surge. CEBPa transcript levels in Molm14 cells (an AML cell line with a FLT3/ITD mutation) rose 3–5-fold over baseline following treatment with AC220. This is consistent with our previously published findings, and suggests at least one mechanism for the observed release of the differentiation block observed in the AC220-treated patients. These clinical and correlative laboratory results suggest that effective, sustained in vivo FLT3 inhibition in AML patients with FLT3/ITD mutations induces terminal differentiation in blasts in many ways similar to that seen with all trans retinoic acid in acute promyelocytic leukemia. Furthermore, these findings demonstrate the direct link between the growth factor receptor pathway and control of differentiation, and provide new insight into mechanisms of leukemogenesis. Disclosures:Levis:Ambit Biosciences, Inc: Consultancy.

Bone Marrow Specific Scatter Pattern on Multiple Myeloma and the M:E Ratio and Cellularity Analysed by the CELL-DYN 4000® Methodology

INTRODUCTION: Optical microscopy is the reference method for the morphological evaluation of bone marrow aspirates; however, in spite of permitting a good qualitative evaluation, the relative quantification of the different type of cells is time consuming and not precise. Regardless the efforts to develop automatic methodologies, no precise and reliable methods are available. CELL-DYN 4000® haematological analyzer uses the MAPSS® technology, based on optical light dispersion, fluorescence and impedance, with great accuracy in differential cellular blood counts.STUDY PURPOSE:to establish the correlation between the optical microscopy and the automatically CELL-DYN 4000® bone marrow examination concerning the cellularity and the myeloid/erythroid ratio.to study the bone marrow scatter pattern on haematological diseases.MATERIAL AND METHODS: 84 bone marrow samples (in K3EDTA) collected for diagnostic purposes among patients attending the Haematology clinic of Centro Hospitalar de Coimbra. Bone marrow films were stained by May-Grünwald-Giemsa's, analysed by two different Haematologists and the diagnosis were confirmed by flow cytometry studies. Bone marrow automatic analysis were performed by CELL-DYN 4000® using CBC(N), Extended Count(W) and Resistant(R) methodologies.Statistical analysis: Pearson's correlation for agreement between automatic methodologies for erythroid lineage and lymphoid series counts; Pearson's correlation for M:E ratio; Cohen's Kappa Test for the cellularity; Fisher's Exact Test to determine whether a specific graphical pattern was associated with a particular haematological disease.RESULTS: Comparing bone marrow studies by manual and automatic methods we verified:a good correlation (r=0,769, p<0,0001) with R methodology for erythroid lineage and lymphoid series, despite the interference of erythroblasts in the lymphoid series;an excellent agreement (k>0,808) in the cellularity assessment and an excellent correlation (r=0,955; p<0,0001) with the R methodology in M:E ratio.We also found a very good association (c2=24; p<0,0001) of a specific scatter pattern and Multiple Myeloma (n=10) which is different from Plasmocytic Leukaemia (n=2) and Monoclonal Gammopathy of Undetermined Significance (MGUS) (n=2).CONCLUSIONS: The present study shows that the bone marrow M:E ratio, as well as the cellularity evaluation by CELL-DYN® 4000 R methodology are reliable. It also shows the association between a specific graphic pattern and Multiple Myeloma. Morphological analysis is the gold standard for bone marrow examination and it will be, probably, never discharged, however, these data confirm the possibilities of having a good global evaluation of bone marrow samples by automatic methods.

Hematologic effects of subcutaneous administration of recombinant human granulocyte colony-stimulating factor (filgrastim) in healthy alpacas

To determine the effects of SC administration of filgrastim on cell counts in venous blood and bone marrow of healthy adult alpacas. 10 healthy alpacas. Alpacas were randomly assigned to receive treatment with filgrastim (5 microg/kg, SC; n=5) or an equivalent volume of physiologic saline (0.9% NaCl) solution (5) once a day for 3 days. Blood samples were obtained via jugular venipuncture 1 day prior to treatment and once a day for 5 days commencing 24 hours after the first dose was administered. Complete blood counts were performed for each blood sample. Bone marrow aspirates were obtained from the sternum of each alpaca 48 hours before the first treatment was administered and 72 hours after the third treatment was administered. Myeloid-to-erythroid cell (M:E) ratio was determined via cytologic evaluation of bone marrow aspirates. In filgrastim-treated alpacas, substantial increases in counts of WBCs and neutrophils were detected within 24 hours after the first dose was administered. Band cell count and percentage significantly increased 24 hours after the second dose. Counts of WBCs, neutrophils, and band cells remained high 48 hours after the third dose. Red blood cell counts and PCV were unaffected. The M:E ratio also increased significantly after treatment with filgrastim. Filgrastim induced rapid and substantial increases in numbers of circulating neutrophils and M:E ratios of bone marrow in healthy alpacas. Therefore, filgrastim may be useful in the treatment of camelids with impaired bone marrow function.