Bone Marrow Trephine Biopsy Specimens Research Articles

The Diagnostic Usefulness of Bone Marrow Examination and Bone Marrow Culture in Patients with Pyrexia of Unknown Origin

Introduction: Bone marrow cultures (BMCs) are usually obtained in the diagnosis of pyrexia of unknown origin (PUO). In the effort to identify the cause of systemic infection, BMCs are often performed in addition with blood cultures (BCs) and cultures of other body fluids and tissues. The practice of performing BMC and Blood cultures is long-standing, but the medical importance is questionable. Given the importance of cost effectiveness, such practices must be assessed on the basis of their cost-benefit to patient outcomes. In this study we compared the value of BMC with BC and studied the usefulness of the histopathological features of bone marrow trephine biopsy specimens in detecting the cause of systemic infections in selected patient population with PUO.
 Objective: To determine the usefulness of bone marrow culture for the diagnosis of different causes of pyrexia of unknown origin.
 Study Design: Descriptive Cross-sectional study
 Place and Duration of Study: This study was conducted at Peoples University of Medical and Health Sciences for Women Nawabshah Pakistan from June January 2020 to January 2021.
 Materials and Methods: A total 150 patients were included in this study who fulfilled the criteria of pyrexia of unknown origin. The bone marrow aspiration, biopsy, and blood cultures specimens were obtained using standard techniques and bone marrow culture send for microbial analysis.
 Results: 1.5% of patients had hematomas, one diagnosed with Behcet disease, other patients with acute leukemia. The bone marrow aspiration results exhibited that 23 patients (15.3%) diagnose with a hemophagocytic syndrome where 3 cases of infection, 3 cases with hematological malignant disorder, and 7 cases with hemophagocytosis, and repercussion of bone marrow aspiration diagnosed 5 acute leukemia and 2 with visceral leishmaniasis. The usefulness of BMB was determined through an odd ratio of few parameters including thrombocytopenia with odd ratio 4.59 and 95% CI (1.07-8.4), anemia odd ratio 3.17, and CI (1.09-12.92) were considered most associated factors for the usefulness of BMB. BMC in 13 (8.66%) patients out of 150 were found with bacterial growth and 2 (1.33%) cases with bacterial growth were observed in blood culture. There was no fungal and mycobacterial growth observed. In the BMC analysis 2 cases of E. coli, 3 cases with Staphylococcus aureus, 2 cases of Enterococcus sps. while 4 cases of Salmonella typhi and 2 cases of Salmonella paratyphi-A were diagnosed in blood culture analysis.
 Conclusion: The obtained data indicate that blood culture did not divulge bacterial growth without the bone marrow culture. Therefore bone marrow culture was found to be an important diagnostic modality and correlation was found for thrombocytopenia and anemia. Bone marrow biopsy is deemed a useful method to diagnose pyrexia of unknown origin.

SYSTEMIC MASTOCYTOSIS: RADIOLOGICAL POINT OF VIEW

Radiological diagnosis of systemic mastocytosis (SM) can be hard to establish. This difficulty is mainly due to the variable radiological features involving many organ systems (e.g., respiratory, cardiovascular, lympho-reticular, digestive systems, and most commonly skin), and above all, to the broad spectrum of skeletal findings. Skeletal involvement is the most common and prominent imaging feature in patients with SM and represents a prognostic factor as it may entail an aggressive course of the disease. Diagnosis, largely established by histological evaluation of a bone marrow trephine biopsy, supplemented by imaging modalities such as radiography, CT, and magnetic resonance imaging, requires a team approach between the hematologist, radiologist, and pathologist. The general radiologist needs to be familiar with the imaging findings because they may be the first to suggest the correct diagnosis. The primary purpose of this review article was to equip clinicians with pertinent radiological semiotics by presenting relevant radiological features that assist early diagnosis and selection of an effective treatment.

Myelodysplastic syndrome: Discordant involvement of the marrow as observed in the bone marrow biopsy sections

Myelodysplastic syndromes (MDS) are a diverse group of hematological disorders that affect the blood and bone marrow. Laboratory diagnosis of MDS is almost exclusively based on the morphology of hematopoietic cells observed in smears of peripheral blood and bone marrow and this depends solely on the presence of dysplastic changes observed in the granuloid, erythroid, and megakaryocytic cells [1,2]. Up until now evaluation of sections of bone marrow trephine biopsy specimen did not play a significant role in its diagnosis.

First report of the unique expression of RECAF (receptor for alfa feto-protein) in adult B-NHL/CLL patients.

BackgroundLymphoproliferative disorders (LPDs) are a heterogeneous group of diseases characterized by an uncontrolled production of monoclonal lymphocytes. RECAF is the receptor for alpha-fetoprotein, which is re-expressed on malignant cells, thus serving as a broad-spectrum tumor marker.MethodsThe current study is a retrospective study carried out on 200 archival bone marrow trephine biopsy specimens [60 normal control (NC), 38 pathological control (PC) and 102 lymphoproliferative diseases (LPD) specimens]. RECAF expression was assessed using immunohistochemistry.ResultsThe percentage of cells that are positive for RECAF was significantly higher in the LPD group than in the NC group (P=0.007), while there was no significant difference between non-Hodgkin lymphoma (NHL) patients and PC regarding the number of RECAF positive cells (P=0.1). RECAF showed a unique expression pattern among the different subtypes of LPD. None of the hairy cell leukemia (HCL) expressed RECAF, while the highest percentage was seen in follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL) (P=0.001). Compared to routine histopathology, RECAF was more sensitive in detecting bone marrow (BM) infiltration in FL, mantle cell lymphoma (MCL), and DLBCL (P=0.01).ConclusionRECAF is significantly expressed in the BM of NHL/chronic lymphocytic leukemia (CLL) patients. RECAF shows a unique expression pattern among the different subtypes of LPD. Furthermore, RECAF may help to detect bone marrow infiltration in lymphoma cells. This may help in the diagnosis, follow-up, and targeting of LPD.

PB1906 FROM ACADEMY TO REAL WORLD: THE GRAY AREA OF THE LARGE GRANULAR LYMPHOCYTES LEUKEMIA

Background:Large granular lymphocytes (LGLs) are defined as large white blood cells (WBC) that are morphologically characterized by an abundant cytoplasm with azurophilic granulation. LGLs belong to CD3+CD56− cytotoxic T cells (CT) or natural killer (NK) cells. The NK cells are roughly defined by a lack of the T cell marker CD3 and a presence of CD56 (or NCAM). LGL leukaemia represents a chronic lymphoproliferative disorder of CTLs, a malignancy that involves lymphocyte infiltration of multiple organs, including the BM, liver, and spleen.Aims:The aim of this retrospective study was to achieve information about LGL and the association between LGL and clinical characteristics.Methods:A retrospective study analyzing the global diagnosis of LGL leukaemia and factors influencing the disease evolution at the district hospital and included cases with the previous or recent diagnosis of LGL leukaemia between January 2015 and December 2018. Special emphasis was taken on the results of bone marrow examination, flow cytometry, immunophenotype, and results of molecular studies to assess for T‐cell receptor (TCR) gene rearrangements. A total of 26 patients were confirmed as having LGL leukaemia, and they fulfilled the WHO classification criteria. May‐Grünwald‐Giemsa–stained peripheral blood smears were examined for the presence of large granular lymphocytes. Bone marrow trephine biopsy and aspirate specimens (when was applicable) were obtained. Bone marrow cellularity and the pattern of lymphocyte infiltration were determined on core biopsy or aspirate clot specimens stained with H&E. Statistic The Fisher exact test was used to analyze differences between the T‐LGL and T‐LGL/other LPD groups. The median time from completion of last treatment with rituximab to the diagnosis of LGL disorder was 30 months.Results:LGL leukaemia or T‐LGL expansion were identified in the global group. Near total of the patient 21 (80.7%) had a direct history of a clonal B‐cell lymphocytosis, monoclonal B‐cell lymphocytosis (MBL), or clonal B‐cell disorder concomitantly with or shortly after the LGL diagnosis. The other predominant diagnosis was rheumatoid conditions in 9 patients (34.6%) (including Felty syndrome) and monoclonal gammopathy of unknown significance (MGUS) 7 patients (26.9%). The median age at the time of diagnosis was 68.5 years (range, 20‐86 years). B‐symptoms were reported by 4 (15.38%) patients, as well as Lymphadenopathy/splenomegaly was identified in 5 (19.23%). 4 individuals received treatment prior to the diagnosis of LGL, and the predominant treatment was methotrexate/rituximab. The favourite regimens in our daily practice were cyclosporin, rituximab or intermittent G‐CSF. The overall response rate at 3 months and complete response rate for the various treatments were as follows: steroids (11% and 3%), methotrexate (55% and 21%), rituximab (64% and 47%), cyclosporine (19% and 3%), respectively. All patients responded to rituximab after methotrexate treatment had failed. The mean number of treatments was 3.4 (range, 1‐5).Summary/Conclusion:Whether or not the T‐LGL and T‐LGL/other LPD groups are correlated with specific clinical‐biological features remains uncertain and is the subject of ongoing research. We don’t perform routinely in our clinical practice an investigation for the Constitutive activation of STAT3. In our series, we have not reported direct LGL‐related deaths.

Lysosomal storage disorders: Morphologic appraisal in Indian population.

Lysosomal storage disorders (LSDs) comprise a group of at least 50 distinct genetic diseases, each one resulting from the deficiency of a particular lysosomal enzyme involved in metabolism. We attempt to study and further subclassify pediatric LSDs into Gaucher's and non-Gaucher's category based on the morphologic variables seen in the bone marrow aspiration smears and trephine biopsy sections. Pediatric (<12 years age) cases of LSDs diagnosed by bone marrow aspiration and trephine biopsy specimens, in the last 12 years period, were retrieved. The archival material and the relevant clinical as well as hematologic parameters were reviewed. From January 1997 to December 2008, 55 cases were diagnosed as LSDs. Based on bone marrow morphology, 56% (n = 31) cases were diagnosed as non-Gaucher's and the remaining 44% (n = 24) cases as Gaucher's disease, the ratio being 1.29:1. Anemia and thrombocytopenia were more commonly observed in Gaucher's disease (91.67 and 62.5%) as compared to non-Gaucher's group (74.19 and 19.35%). Neurologic symptoms and signs were more frequently present in non-Gaucher's cases (45.16%) as compared to Gaucher's group (29.17%). LSDs can be classified into Gaucher's and non-Gaucher's subtypes based on the characteristic cytomorphology of the storage cells in Giemsa-stained bone marrow aspiration smears and on hematoxylin and eosin-stained trephine biopsy sections. This approach would be fairly adequate for therapeutic and prognostic purposes in resource.constrained settings, where enzyme studies and mutational analysis may not be easily available.

Diagnosis of latent polycythemia vera: A clinician's opinion

to identify the clinical features of latent polycythemia vera (PV) as an independent nosological entity. The investigation enrolled 81 patients (50 with extensive (manifest) PV and 31 with latent PV) who had visited the Outpatient Department, Hematology Research Center, Ministry of Health of Russia, in 2014 to October 2015. The gender distribution of the patients was statistically comparable in the analyzed groups. The patients with manifest PV were slightly older than those with latent PV: the median age in the compared groups was 56 and 44 years, respectively. Red blood cell counts, hemoglobin concentrations, and packed cell volume were higher in the patients with manifest PV. Blood platelet counts were higher in the latent PV group. There were no differences in the number of white blood cells in the compared groups. All the patients were JAK2 V617F mutation carriers. The JAK2 allele load was significantly higher in the manifest PV group than in the latent PV group. The compared patient groups differed in the rate of thromboses in the history or at diagnosis. In the patients with latent PV, thromboses were detected in 38% of cases versus 16% in those with manifest PV. In latent PV, there were mainly venous thromboses; abdominal vascular thromboses were diagnosed with a high frequency. Arterial thromboses were revealed in only 2 cases. Chronic myeloproliferative disease that is characterized by the JAK2 V617F mutation, borderline hemoglobin counts, and morphological features of a bone marrow trephine biopsy specimen, which are specific for PV, is an independent PV variant, namely: latent PV.

Diagnostic and Prognostic Relevance of Bone Marrow Microenvironment Components in Non Hodgkin’s Lymphoma Cases Before and After Therapy

Objective:To evaluate stromal cells of the bone marrow microenvironment (BMM) in bone marrow trephine biopsy (BMTB) specimens, with a focus on fibronectin, tumor necrosis factor- alpha (TNF-α) and L-selectin in Non-Hodgkin’s lymphoma (NHL) patients, before and after therapy.Materials and Methods:A total of 80 de novo NHL patients, 64 with B-cell lymphomas 80%, (follicular cell lymphoma (FCL) in 32, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) in 12, and diffuse large cell lymphoma in 20) and 16 with T-cell lymphomas (20%) all diagnosed as T-Lymphoblastic lymphomas, were evaluated before and after therapy. For comparison, 25 age and sex matched BM donors, were included as a control group. BMTB material and BM aspirates were taken for morphological assessment of stromal cells, the plasma of these samples being examined for TNFα and L-selectin by ELISA, and fibronectin by radial immunodiffusion (RID).Results:BM stromal cells comprising reticular macrophages and fibroblasts were elevated in 53.3% of NHL cases at diagnosis, while BM fibronectin levels were decreased and BM TNFα and L-selectin were higher than in controls (p<0.05). In NHL cases, elevated values of BM TNFα and BM L-selectin were associated with signs of aggressive disease, including >1 extra nodal sites, detectable B symptoms, high grade, BM and CNS invasion, and a high International prognostic index (IPI) (p<0.05).Conclusion:BMM components, TNFα, L-selectin and fibronectin, in NHL can be useful in evaluating disease activity, extent and response to treatment and as prognostic markers according to the IPI.

Multiple Myeloma and Bone Marrow Microenvironment Immunohistochemical Study of the Expression of 15 Proteins Related to Myeloma Bone Disease

Objective: Neoplastic milieu is an integral part of all malignant diseases including multiple myeloma and plays variable role in their development, retention/adhesivity, resistency or sensitivity to therapeutic approach, homing and also paraneoplastic manifestations. Relatively genetically stable milieu may play an important role in new specific molecular therapeutic approaches and therefore should be contextually studied with neoplastic cells as complex neoplastic tissues. The expressions of 15 proteins with close relation to the development of myeloma bone disease (MBD) were analysed in consecutive multiple myeloma specimens.Methods: Bone marrow trephine biopsy specimens (n=57) with multiple myeloma were included in our prospective study. FFPE tissues were processed in app. 5microm sections and placed on charged slides. The indirect immunohistochemical staining was applicated after antigen retrieval and commercial primary antibodies were used for the detection of observed proteins. Standard secondary antibody and ABC method were included in visualisation. We analysed the expressions of MIP1alfa, Annexin A2, TRAP, DKK-1, RANK, RANKL, OPG, Sclerostin, Activin A, NFkappaB proteins (p50, p52, p65), p62 (sequestosome 1), MMP9 and RUNX2.Results: Bone marrow multiple myeloma specimens showed variable positivity of MIP1alfa in 60% (cut-off point 20%), Annexin A2 in 42% (myeloma cells, cut-off point 30%) and in 74% (stromal cells, cut-off point 5%), TRAP in 28% (cut-off point 5%), DKK-1 in 23% (cut-off point 30%), RANK in 53% (cut-off point 30%), RANKL in 70%, OPG in 39% (cut-off point 5%), Sclerostin in 95% (cut-off point 90%), Activin A in 35% (cut-off point 30%), cytoplasmic positivity of p50 in 5% (cut-off point 10%), p52 in 86% (cut-off point 10%), p62 in 91% (cut-off point 10%), p65 in 89% (cut-off point 10%), positivity of MMP9 in 22% (cut-off point 30%) and positivity of RUNX2 in 56% (cut-off point 30%).Conclusion: Our study showed variable expression of proteins related to MBD in multiple myeloma and its bone marrow microenvironment that imply biological heterogeneity, different development and stromal plasticity in this complex hemato-oncological disease. The exact and contextual knowledge of the engaged signaling pathways may suggest more specific or tailored therapeutic approaches (e.g. anti-RANKL, anti-DKK-1, anti-Sclerostin, anti-Activin A).Supported by the grant NT 14393. DisclosuresNo relevant conflicts of interest to declare.

Bone marrow aspirate microscopy v. bone marrow trephine biopsy microscopy for detection of Mycobacterium tuberculosis infection.

Tuberculosis (TB) remains a global health problem. According to the 2013 Global Report on Tuberculosis, 8.6 million people developed TB in 2012 and 1.3 million died from the disease. An estimated 13% of people who developed TB in 2012 were HIV-positive, and 75% of these lived in Africa. While pulmonary TB is the commonest form of Mycobacterium tuberculosis infection, extrapulmonary TB is increasingly being detected in HIV-positive patients. Definitive diagnosis of disseminated TB is a challenge owing to atypical presentations and diagnostic difficulties (negative chest radiograph and sputum microscopy and culture). A rapid diagnosis of disseminated TB is desirable, as early initiation of treatment can reduce mortality. Although TB culture is the gold standard for diagnosis of TB, it has a long turnaround time (up to 6 weeks). To identify a potentially faster and more effective diagnostic strategy for disseminated TB. A retrospective 18-month review, conducted at a tertiary hospital, comparing histological findings of an auramine O-stained bone marrow aspiration (BMA) smear and a bone marrow trephine (BMT) biopsy specimen with the gold standard of TB culture. Microscopic examination of BMA smears and BMT biopsy specimens offers a rapid diagnostic strategy, with results available on the same day for the former and within 4 days for the latter. BMT histological examination had a significantly higher detection rate than BMA auramine O staining compared with TB culture. We recommend that BMT biopsies remain an essential part of the diagnostic work-up for disseminated TB.

Lymphoplasmacytic lymphoma and marginal zone lymphoma in the bone marrow: paratrabecular involvement as an important distinguishing feature.

The differential diagnosis between bone marrow involvement by lymphoplasmacytic lymphoma (LPL) and marginal zone lymphoma (MZL) is challenging because histology and immunophenotype of both diseases overlap. We revisited the diagnostic pathology features of both diseases in the bone marrow. We studied a series of bone marrow trephine biopsy specimens from 59 patients with Waldenström macroglobulinemia without extramedullary involvement and bone marrow biopsy specimens from 23 patients with well-characterized MZL who also had bone marrow involvement. H&E- and immunoperoxidase-stained sections of bone marrow trephine biopsy specimens as well as flow cytometry and classic cytogenetics performed on aspirations were reviewed. The study was complemented with MYD88 L265P mutation analysis of all samples. The most distinguishing features of LPL with respect to MZL were focal paratrabecular involvement (P < .001), the presence of lymphoplasmacytoid cells (P < .001) and Dutcher bodies (P < .001), increased numbers of mast cells (P < .001), and the MYD88 L265P mutation (P < .001). LPL can be reliably distinguished from MZL in the bone marrow by using a combination of pathology characteristics. Our findings stress the diagnostic importance of using the combination of the following parameters for a correct LPL diagnosis: paratrabecular infiltration, the presence of lymphoplasmacytoid cells and cells with Dutcher bodies, and an increased number of mast cells in addition to the presence of MYD88 mutation.

Molecular analysis of immunoglobulin genes in the tumor B cells in splenic marginal zone lymphoma

To determine the immunoglobulin variable heavy chain (gVH) genes and their somatic mutations and to compare these data with the clinical and laboratory parameters of patients and the outcomes of the disease. The investigation enrolled 24 patients (9 men and 15 women whose age was 32 to 77 years (median age, 60 years) with splenic marginal zone B-cell lymphoma (SMZBCL). The latter was diagnosed on the basis of histological and immunohistochemical examinations of a bone marrow trephine biopsy specimen, immunophenotyping of peripheral lymphoid cells or bone marrow aspirates. The mutational status of the IgVH genes was analyzed in all the 24 patients. It was found that the tumor cells contained mutated IgVH genes (the VH1-family genes participated in most cases) in 15 (62.5%) patients with SMZBCL and unmutated ones in 9 (37.5%). There was a tendency towards more common tumor progression in patients with unmutated IgVH genes than in those with mutated ones. The presence of SMZBCL cases with both mutated and unmutated IgVH genes and the higher frequency of the VH1-2 gene are likely to indicate the molecular heterogeneity of the origin of this lymphoma.

The value of anti-Pax-5 immunostaining in pediatric acute leukemia

Background Pax-5 protein is a transcription factor expressed in the B-lymphoid lineage from the earliest detectable precursor to the mature B-cell stage. Its expression is closely correlated with B-cell precursor acute lymphoblastic leukemia (pre-B-ALL), which is one of the most common childhood malignancies. Aim of the work To evaluate the expression of anti-Pax-5 monoclonal antibody on leukemic blasts in pediatric patients with acute leukemia and to compare it with anti-CD20 to show its diagnostic utility. Patients and methods This study was carried out on 35 pediatric patients diagnosed with acute leukemia, who were divided into two groups: Pre-B-ALL and Acute, myeloid leukemia (AML). All patients were subjected to a complete assessment of history, full clinical and laboratory examination, bone marrow aspirate and biopsy, and immunophenotyping. Immunostaining of bone marrow trephine biopsy specimens using Pax-5 and CD20 monoclonals was carried out for all cases. Results Pax-5 was positive in 79.1% of cases in the pre-B-ALL group and negative in all AML patients. CD20 was positive in 37.5 and 18% of cases in the ALL and the AML groups, respectively. Pax-5 appeared to be more sensitive (79.2%) and more specific (100%) than CD20 in differentiating ALL and AML. Conclusion Our results highlight the beneficial role of including Pax-5 in an immunohistochemical panel to diagnose acute leukemia of B-cell lineage.

Intracellular and extracellular immunoglobulin crystals assuming a multitude of geometric shapes in an IgG‐λ plasma cell neoplasm

Intracytoplasmic immunoglobulin deposition is frequently associated with plasma cell neoplasms but generally takes on a globular form (Russell bodies). The presence of crystalline immunoglobulin structures within malignant plasma cells occurs rarely 1-7. Here we show images of a post-treatment bone marrow biopsy from a 47-year-old woman diagnosed with a plasma cell neoplasm based on the presence of a serum monoclonal IgG-λ paraprotein (0.5 g/dL) and 30% plasma cells with cytoplasmic λ light chain restriction demonstrated by flow cytometry 8. Although she initially achieved a complete remission with intensive therapy, this bone marrow biopsy shows early progression of disease with 10–15% monoclonal λ light chain-restricted plasma cells. Additionally, focal areas containing large, rhomboidal-, hexagonal-, rectangular-, and square-shaped crystalline structures were noted in both the bone marrow aspirate (Image 1A, Wright-Geimsa stain, arrowheads) and trephine biopsy specimens [Image 1B, hematoxylin & eosin (H&E) stain]. Scattered lymphohistiocytic aggregates infiltrated by eosinophils and occasional multinucleated giant cells were present (Image 1C and inset, H&E). Some of the crystals are clearly extracellular (Image 1C, arrows) while others appear to be intracytoplasmic as suggested by flattening of the nuclei in cells juxtaposed to crystals (Image 1C, arrowheads). An immunohistochemical stain for CD138 highlights plasma cells (Image 1D) and outlines the intracellular subset of crystals. Stains for κ and λ light chains show λ light chain restriction, and the crystals are similarly outlined by the λ (Image 1E), but not the κ, stain (Image 1F). Immunostaining for CD68 delineated macrophages and giant cells but did not outline the crystalloid structures, thus supporting the plasma cell localization of the crystals. Staining for acid-fast bacteria and fungus was negative throughout, and a congo red stain did not highlight any amyloid deposits nor did it mark the crystals. Of note, the patient's CBC showed no cytopenias at this time, though there was recurrence of the serum monoclonal paraprotein (0.86 g/dL IgG-λ). She remains alive with a normal CBC and stable marrow disease 18 months after high-dose therapy. Although crystal deposition within plasma cells has been reported in reactive conditions, including rheumatologic disorders and helicobacter-associated gastritis, the monoclonal nature of the immunoglobulin in this case as demonstrated by both flow cytometry and immunohistochemical analysis argues against a reactive process. Crystal storing histiocytosis (CSH), in which immunoglobulin crystals are deposited in macrophages is an unusual variant of this phenomenon and is also associated with plasma cell and other lymphoproliferative neoplasms 9. The κ light chains predominate in CSH, as they appear to be more resistant to degradation by macrophage lysosomal enzymes than λ light chains. The mechanism underlying the accumulation of immunoglobulin crystals within neoplastic plasma cells as presented here is not certain but does not appear to favor either κ or λ light chains and may involve abnormalities in protein synthetic and transport pathways. The significance of immunoglobulin crystals in plasma cell myeloma is unknown, but it has been hypothesized that their presence may portend a nonprogressive clinical course and therefore a favorable prognosis 10.

Ultrasound: a new tool for precisely locating posterior iliac crests to obtain adequate bone marrow trephine biopsy specimen

Ultrasound imaging has evolved as a very useful tool in medical science. However, the technique of ultrasound-guided bone marrow trephine biopsy (BMTB) has not been routinely used by haematologists and...

Fluorescence in situ hybridisation analysis of bone marrow trephine biopsy specimens; an additional tool in the diagnostic armoury

Fluorescence in situ hybridisation (FISH) analysis is now widely employed in the diagnosis and risk stratification of a wide range of malignant diseases. While this technique is used successfully with...

Immunophenotyping of Chronic B-Cell Neoplasms: Flow Cytometry versus Immunohistochemistry

Morphological differentiation between benign and malignant lymphoproliferative disorders (LPDs) can be challenging. Immunophenotyping (IPT) by either technique, flow cytometry or immunohistochemistry (IHC), is an important step in solving such difficulty. Thirty-five newly diagnosed patients with chronic B-cell neoplasms (11 chronic lymphocytic leukemia, 22 non Hodgkin lymphoma and 2 hairy cell leukemia) were included in this study with age range from 20 to 70 years. Monoclonal antibodies surface expression using lymphoproliferative disorders panel (CD45, CD19, CD5, CD10, CD11c, CD20, CD22, CD23, CD38, CD79b, FMC7, CD103, CD25, kappa and lambda light chains) by flow cytometry was done on bone marrow samples. CD20, CD5, CD23, Bcl-2, Bcl-6, kappa and lambda light chain immunostaining were performed on fixed bone marrow trephine biopsy specimen. The sensitivity of IHC was 81.8% in chronic lymphocytic leukemia (CLL) and 100% in non Hodgkin lymphoma (NHL) as regards CD20, 100% in both groups as regards CD5, 46% in CLL and 66.7% in NHL as regards CD23, 33.3% in CLL and 50% in NHL as regards kappa chain, 20% in CLL and 33.3% in NHL as regards lambda chain. We found that IHC and flow cytometry are equally effective in diagnosing CLL; however, IHC might be slightly more sensitive than flow cytometry in detecting bone marrow infiltration in NHL and hairy cell leukemia (HCL).

PTPIP51 is phosphorylated by Lyn and c-Src kinases lacking dephosphorylation by PTP1B in acute myeloid leukemia

Protein tyrosine phosphatase interacting protein 51 (PTPIP51) is known to be expressed in blood cells with restriction to the myeloid lineage. All myeloid progenitor cells are PTPIP51 positive except for the myeloblasts. To define the expression of PTPIP51 in acute myeloid leukemia (AML), we performed immunohistochemical experiments with peptide specific antibodies (C-terminus, N-terminus and aas 114–129) to PTPIP51 with samples of AML bone marrow trephine biopsy specimens. AML blasts reacted positive for PTPIP51 protein encompassing the C-terminal sequence. Healthy bone marrow displayed an exclusive staining for the N-terminal containing form of PTPIP51. Moreover, PTPIP51 protein was highly phosphorylated at its tyrosine 176 residue. Acquired confocal images of AML cells displayed an absence of PTP1B and revealed a co-localization of PTPIP51 and Lyn. Duolink proximity ligation assays (DPLA) corroborated an interaction for PTPIP51 with Lyn and c-Src. In AML blasts rarely an interaction of PTPIP51 with PTP1B and Raf-1 was seen. Furthermore, DPLA signals were also obtained for PTPIP51 and c-Kit in AML cells. Therefore, PTPIP51 was identified as a new signal molecule of the c-Kit signaling pathway. By the phosphorylation done by Lyn, c-Src and c-Kit, PTPIP51 is prevented to influence mitogen activated protein kinase pathway on Raf-1 level contributing to increased proliferation of AML cells.

Elemental Arsenic Levels in Plasma, Cellular and Tissue Compartments After Oral Arsenic Trioxide Maintenance Treatment: Implications On Long-Term Safety.

Abstract 2064Poster Board II-41 Background.Arsenic trioxide (As2O3) is a standard medication in the management algorithm of patients with acute promyelocytic leukemia (APL). With the advent of oral As2O3,outpatient treatment, convenient dosing and long-term post-remission maintenance therapy have become possible. The pharmacokinetics, efficacy and safety profile of short-term oral As2O3therapy are well-defined. With prolonged oral-As2O3 therapy, possible accumulation of elemental arsenic and its possible long-term side effects must be determined. Materials and methods.Paired peripheral blood (PB) and bone marrow (BM) (aspirate and trephine biopsy) specimens were prospectively collected as part of an oral-As2O3 treatment protocol of patients. The samples were separated into plasma (P), white blood cell (WBC) and red blood cell (RBC) fractions. Elemental arsenic was measured by inductively coupled plasma-mass spectrometry (ICP-MS, ELAN DRCplus 6100, PerkinElmer-SCIEX, Canada) after dilution with 2% nitric acid +/– 3% methanol. Bone marrow trephine biopsies were dried at 65°C overnight, completely digested with acid, and diluted to appropriate concentrations. Results.There were 15 male and 11 female APL patients. Seven WBC specimens could not be analyzed due to inadequate material. Specimens were collected at 1 month (Group 1), 2–12 months (Group 2) and 24–41 months (Group 3) after cessation of oral-As2O3 treatment. The median cumulative dose of oral-As2O3 was 1980 (560–3680) mg. As control, samples were obtained from 12 anonymous individuals, whose specimens were collected as part of their diagnostic evaluations. These controls had no known medicinal exposure to arsenic. The mean elemental arsenic levels in the specimens were shown below.Total doseMean elemental arsenic concentrations (nM/L)GroupsAs2O3PB–PPB–WBCPB–RBCBM–PBM–WBCBM–RBCBONEControl (n=12)0 mg80.9112.9 (n=11)120.986.6123.1126.20.057 mg/g1 (n=8)1399 mg302.6286.2303.1320.4302.6 (n=6)320.50.127 mg/g2 (n=7)2461 mg99.4130.0 (n=6)171.388.4155.8 (n=5)179.30.080 mg/g3 (n=11)1727 mg112.0161.9172.0120.6106.6 (n=10)171.40.073 mg/gElemental arsenic levels in group 1 were significantly higher than groups 2 and 3 in all specimens tested. However, groups 2 and 3 were comparable to controls, indicating that arsenic was rapidly excreted once oral-As2O3 treatment was stopped. In group 1 patients, all specimens tested showed comparable arsenic levels. In group 2 patients, arsenic levels were significantly higher in BM–WBC (but not PB-WBC) as compared with PB-P and BM-P (p=0.043 for both). In group 3 patients, all cellular specimens tested showed comparable arsenic levels to plasma levels. Conclusion. Immediately after cessation of treatment when plasma arsenic level was still high, elemental arsenic was distributed comparably in different compartments. However, when plasma arsenic levels started to decline, preferential concentration of arsenic occurred in the cellular compartments. This could be related to transfer of arsenic in bones to the developing hematopoietic cells in the marrow milieu. Finally, two or more years after cessation of As2O3 therapy, elemental arsenic levels returned to control levels; a reassuring observation that long-term arsenic accumulation did not take place when oral-As2O3 was used therapeutically during a finite period of time. Disclosures:Off Label Use: Oral arsenic trioxide is a novel preparation or arsenic trioxide currently under consideration for registration in Hong Kong for APL treatment. The University of Hong Kong holds the patent for the product.

Immunophenotyping of acute lymphoblastic leukemia using immunohistochemistry in bone marrow biopsy specimens

Flow cytometry is the preferred method of diagnosing and immunophenotyping acute lymphoblastic leukemia (ALL). However, there are situations in which immunohistochemical staining (IH) of bone marrow trephine biopsy specimens can be used to provide immunophenotypic information. To evaluate the use of IH and to confirm its value in diagnosing and typing of ALL, we studied 50 cases of denovo ALL that were previously classified into pre B, T and B by morphologic, cytochemical and FC methods. Paraffin embedded bone marrow trephine biopsies sections were stained using a panel of antibodies,namely, myeloperoxidase (MPO), terminal deoxynucleotidyl transferase (TdT), CD10, CD20, CD79a, CD3. The cases included 37 pre BALL, 10 T ALL and 3 mature BALL. TdT was the most commonly expressed antibody and was positive in 41 of 50 cases of ALL (82%) and in 95% of pre B ALL cases. CD79a and CD10 were positive in 68% and 65% of pre B ALL cases, respectively. CD79a showed similar positivity in B ALL cases (66%). CD 20 was positive in 66% of mature B ALL cases but less positive in pre B ALL (22%). CD3 was positive in 70% of T ALL cases and negative in other ALL subtypes. All of the cases were negative for MPO. Diagnosis and immunophenotyping of acute lymphoblastic leukemia is possible using immunohistochemical staining of bone marrow trephine biopsies.