Usefulness of Unicel DxH 800 Cell Population Data in the Detection of Plasmacell Leukemia: 2 Cases Report

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4915-4915
Author(s):  
Donatella Raspadori ◽  
Santina Sirianni ◽  
Alessandro Gozzetti ◽  
Francesco Lauria ◽  
Claudio Fogli ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Population ◽  
Blood Smear ◽  
Normal Values ◽  
Population Data ◽  
Morphological Features ◽  
Short Report ◽  
Lymphocyte Population ◽  
Literature Report

Abstract Abstract 4915 Introduction and methods. Lymphoproliferative disorders (LD) are characterized and described by lymphocyte population with heterogeneous morphological features both in optical microscopy revision and in flow cytometry. Several literature report the clinical usefulness of Cell Population Data (CPD) provided by Beckman Coulter hematology analyzers. Abnormal values of CPD correlate with morphological abnormalities of leukocytes. In this work we present a case report of a plasmacell leukemia analyzed with UniCel DxH800 device. DxH800 performs leukocytes differential with the Flow Cytometric Digital Morphology (FCDM) technology, based on the measurements of Volume (V), Conductivity (C) and 5-angle Scatter light laser (MALS, UMALS, LMALS, LALS, AL2) on cells in their native state. Mean and standard deviation of FCDM measurements are collected in 56 CPD. Normal CPD values were computed from a 42 normal samples. Results. A 47-years old woman, referring continuous asthenia, was addressed to our lab with clinical suspect of LD with leukocytosis (WBC=17190/μl, LY#=3800). DxH800 analysis confirmed WBC count adding some important comments. WBC histogram showed a big peak in lymphocyte population. Differential values reported neutrophilia and lymphocytosis while scatterplot showed a lymphocyte cluster very close to the neutrophil one. CPD suggested a heterogeneous neutrophil population with low volume and low scatters (MALS, UMALS, LMALS, LALS, AL2 in arbitrary units) respectively of 106, 90, 112, 62, 75 vs normal values of 144, 137, 143, 158, 159. Examination of blood smear showed a lot of lymphocyte with nuclear immaturity and plasmoblast features. Immunophenotype revealed that 63% of the WBC were CD138+/CD38+, CD56+ CD200-, CD27- CD20-. Bone marrow biopsy confirmed the plasmacell leukemia diagnosis. A 65-years old man was admitted to our department for a light lymphocytosis associated with a IgGk monoclonal component. Immunophenotipic analysis showed a NK proliferation (CD3 50%, CD4 38%, CD8 34%, CD2 92%, CD7 92%, CD16 45%, CD56 48%, CD57 54%). DxH800 analysis reported LY#=3.6/μl and MO#=1,6/μl. LY CPD indicate cells with light signals of degranulation (MALS=56, UMALS=60, LMALS=63 vs normal values of 66, 60 and 63 ) together with abnormal monocyte CPD such as MV=157, MC=136, MALS=79, UMALS=80, LALS=75 vs normal values of 164, 129, 85, 80 and 75 respectively. All this data induced us to look for a mononuclear population different both from lymphocytes and monocytes in the peripheral blood smear. Bone marrow microscopy analysis showed morphologically abnormal cells that were classified as plasmacells after immunophenotyping (CD138+/CD38+, CD56+, CD45-, CD117+, CD20-, CD27-, CD200+. Further immunophenotypic analysis showed in PB 14% of plasmacells CD138+/CD38+/CD45-. Conclusion. We presented 2 cases report of a plasmacell leukemia whose diagnosis were supported by the useful information of the CPD provided by DxH 800. CPD abnormal values for lymphocytes and monocytes were known to correlate with morphological abnormalites of the cells. For this reason we were triggered to deeply investigate the blood smear of the two patient and we performed the immunophenotyping. This short report confirm the usefulness of CPD provided by UniCel DxH800 as the first check point for the diagnostic route. Moreover we confirm that morphological features in the PB smear discovered during the diagnosis, supported by flow-cytometry data, were properly correlated with CPD values. Disclosures: Fogli: Instrumentation Laboratory: Employment. Di Gaetano:Instrumentation Laboratory: Employment.

Comparison Of Lymphocytic Immune Profiles Of Aplastic Anemia and Hypocellular Myelodysplastic Syndrome

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3719-3719
Author(s):  
Jeffrey J. Pu ◽  
Guillermo Rangel Rivera ◽  
Abigail Sido ◽  
Arthur Berg ◽  
Cinda Boyer ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Myelodysplastic Syndrome ◽  
Aplastic Anemia ◽  
Cell Population ◽  
Bone Marrow Failure ◽  
Expression Patterns ◽  
Lymphocyte Population ◽  
Cd19 Expression

Abstract Background Aplastic anemia (AA) and hypocellular myelodysplastic syndrome (MDS) are two common acquired bone marrow failure diseases. AA is mostly an acquired bone marrow disease caused by cellular and humoral mediated immune attack of hematopoietic stem cells (HSC) due to dysregulation of lymphocytic system, which leads to hematopoietic progenitor cell apoptosis and bone marrow failure. MDS is a group of heterogeneous acquired clonal HSC disorders with ineffective hematopoiesis. Approximately 10% to 20% of MDS manifests a reduced bone marrow cellularity, which comprises hypocellular MDS. There is increasing experimental and clinical indication that an immune-mediated damage to hematopoietic HSCs and changes in the hematopoiesis-supporting microenvironment contribute to the pathogenesis of hypocellular MDS. Because of the similarity of their bone marrow manifestation, hypocellular MDS and AA are often hard to distinguish. Mounting evidence indicates that abnormal activation of cytotoxic T cells plays a crucial role in the pathophysiology of these diseases. One study showed that AA patients have an abnormally activated subpopulation of CD4+ helper cells and a decreased number and function of T regulatory cells in the bone marrow. GVHD mouse models further demonstrated that self-reactive T cells were capable of recognizing non-polymorphic tissue or commensally-derived antigens. Recent literature suggests that immune dysregulation plays a major role in pathogenesis of acquired bone marrow failure disease. However immune profiles of these two diseases have not been thoroughly studied, specially the role of B lymphocyte population. Our study aims to find lymphocytic surface marker expression patterns of hypocellular MDS and AA in both immature cell and lymphocyte populations. Methods This retrospective study analyzed flow cytometry lymphocytic antigen expression profiles from patients diagnosed as AA and hypocellular MDS as per standard criteria. A total of 31 AA and 26 hypocellular MDS patient cases were recruited. The bone marrow aspirate/biopsy data, bone marrow aspiration flow cytometry reports, and Complete Blood Counts (CBC)s from individual patients were analyzed. Using side scatter (SSC) vs. CD45 gating flow cytometry panels, we identified immature cell population (SSClow/CD45low) and lymphocyte population (SSClow/CD45high). We then quantitatively analyzed the expression patterns of 33 cluster differentiation (CD) molecules on individual sample. Finally, we compared the CD expression patterns between AA and hypocellular MDS in both cell populations respectively. Results CD19 expression was significantly higher in AA than in hypocellular MDS in both SSClow/CD45low cell population (P=0.001) and SSClow/CD45high cell population (P=0.003). Hypocellular MDS contains significantly higher CD34high cells than AA in SSClow/CD45low populations (mean:28.5% vs 8.5%; range; 1% to 94% vs 2% to 27%; P=0.04). However, patients with both diseases similarly contains very few CD34high cells in SSClow/CD45high cell population (mean: 0.6% vs 2.6%; range: 0.0% to 2% vs 0.0% to 32%; P=0.99). Conclusion 1. In AA, B cells are highly proliferative in both immature stage and mature stage. This data indicates that B cells which may play a unique role in AA pathogenesis but not in hypocellular MDS. 2. In both AA and hypocellular MDS, the majority of lymphocyte population are mature cells. This data suggests that the pathogeneses of both diseases caused by a persistently dysregulated immune microenvironment, not by an acute insult. CD19 expression pattern may be a useful marker to distinguish AA and hypocellular MDS. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Total Cell Counts of the Bone Marrow of Normal Albino Rats from 1 to 50 Weeks of Age

Blood ◽  
1959 ◽  
Vol 14 (4) ◽  
pp. 409-414 ◽  
Author(s):  
WILLIAM T. BURKE ◽  
CHARLES HARRIS
Keyword(s):  
Bone Marrow ◽  
Cell Population ◽  
Normal Values ◽  
Age Groups ◽  
Considerable Change ◽  
Total Cell ◽  
Albino Rats ◽  
Cell Counts ◽  
Total Cell Population ◽  
Femoral Bone Marrow

Abstract A method is described by which the total nucleated cell count of femoral bone marrow of the rat can be estimated and cell population expressed in terms of differential counts. Normal values of total nucleated cell counts and the cellular distributions are given for seven age groups. These data indicate considerable change in bone marrow total cell population in rats one to 10 weeks of age.

Circulating Megakaryocyte-Derived Cells Detected by Flow Cytometry As Marker of Aggressive Neoplasms of Megakaryocytic Lineage in Acute Megakaryoblastic Leukemia and Allied Malignancies Presenting As Primary Myelofibrosis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1461-1461
Author(s):  
Serena Marotta ◽  
Giovanna Giagnuolo ◽  
Giulia Scalia ◽  
Maddalena Raia ◽  
Santina Basile ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Differential Diagnosis ◽  
Cell Population ◽  
Peripheral Blood ◽  
Primary Myelofibrosis ◽  
Giant Platelets ◽  
Megakaryoblastic Leukemia ◽  
History Of ◽  
Blast Cells

Abstract Abstract 1461 The differential diagnosis of myelofibrotic disorders encompasses chronic primary myelofibrosis (PMF), myelodysplastic syndromes with fibrosis (MDS-F), acute panmyelosis with myelofibrosis (APMF) and acute megakaryoblastic leukemia (AMKL). Most of these conditions are recognized as distinct entities by the WHO 2008 revised classification of myeloid neoplasms; however, the WHO admits that often a definitive diagnosis is problematic, mostly because of specimens with insufficient cellularity (e.g., “dry tap”). Nevertheless, the correct identification of the most aggressive fibrotic disorders (APMF and AMKL) remains crucial, given their poor prognosis and subsequent need of intensive treatment (including transplantation). Even the most recent molecular studies did not result in any contribution in the differential diagnosis. Here we report our experience on a cohort of about 300 patients who were admitted in our bone marrow failure unit because of cytopenia in the last 7 years. All these patients were evaluated by standard peripheral blood and bone marrow cytology, karyotype analysis and bone marrow thephine biopsy, aiming to a definitive hematological diagnosis. Flow cytometry analysis was performed at initial presentation and then serially during the follow up on both peripheral blood and bone marrow aspirate. All patients were classified according to the WHO 2008 revised classification of myeloid neoplasms, and received the best standard treatment based on the specific disease, age and comorbidities. This report focuses on 8 patients who shared a unique flow cytometry finding of an aberrant megakaryocyte-derived cell population, which seems associated with a distinct disease evolution. Two of these patients received the diagnosis of AMKL according to bone marrow aspirate and trephine biopsy; the karyotype was complex in one case (monosomal karyotype, including a 5q-), whereas no Jak-2 mutation or any other genetic lesions could be demonstrated. Their blast cells were CD34+, CD38+, CD45+, CD117+, CD33+, CD13+; in addition, in the peripheral blood, we detected the presence of an aberrant cell population which was CD45-, CD42b+ (CD34+ in one case and CD34- in the other one). In the blood smear, we observed megakaryocyte fragments which likely correspond to this aberrant cell population, as identified by flow cytometry. Other three patients presented with a severe pancytopenia: all of them had a dry tap, and their trephine biopsies documented a massive fibrosis. They had no previous hematological disorder (one suffered from Behcet syndrome), normal karyotype and absence of any typical genetic lesion (i.e., wild-type Jak-2). All of them did not show splenomegaly, increased LDH or leukoerythroblastosis; their peripheral blood smear showed abnormal giant platelets, often resembling megakaryocyte fragments. Flow cytometry documented in the peripheral blood the presence of a distinct population of CD45-, CD42b+, CD61+ cells, which was also CD34+ in one case. These 3 patients were initially classified as PMF, even if APMF could not be ruled out; however, within 6 months they all progressed to AMKL. At this stage, typical CD34+, CD45+ blast cells were accompanied by a progressive increase of CD45+, CD42b+, CD61+ cells. This aberrant megakaryocyte-derived cell population (which could not be demonstrated in patients with thrombocytopenia) was also identified in 3 additional patients, who have a previous history of hematologic disorders: two had a history of pure red cell aplasia (successfully treated by immunosuppressive therapy), and one a 5q- melodysplastic syndrome (responding to lenalidomide, even with transient cytogenetic remission). In all of them we observed the appearance of CD45-, CD42b+ cells in the peripheral blood, which appeared as giant platelets/megakaryocyte fragments in the blood film; this finding within a few weeks was followed by progression to AMKL (5q- was detected in 2 of 3 cases). In conclusion, we demonstrate that aberrant circulating megakaryocyte-derived cells detected by flow cytometry may be useful in the differential diagnosis of myelofibrotic disorders. These giant platelets or megakaryocyte fragments, regardless the initial diagnosis, were associated with early evolution into AMKL, likely representing a surrogate marker for aggressive neoplasms of the megakaryocytic lineage. Disclosures: Risitano: Alexion: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Pluripotent Fibrosis Initiating Mesenchymal Stromal Cell Linked To Cytopenias In LGL Leukemia Through Genetic Reprogramming

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1218-1218
Author(s):  
Pearlie K. Epling-Burnette ◽  
Adam W. Mailloux ◽  
Ling Zhang ◽  
Lynn C. Moscinski ◽  
Sheng Wei ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Microarray Analysis ◽  
Cell Population ◽  
Collagen Type ◽  
Type I ◽  
Bone Marrow Fibrosis ◽  
Control Mscs ◽  
Marrow Fibrosis ◽  
Lgl Leukemia

Abstract Background Large Granular Lymphocyte (LGL) leukemia is associated with chronic lymphoproliferation in association with unexplained cytopenias. Discovery of somatic mutations in STAT3 provides new insights into the molecular basis of LGL leukemia, but the precise mechanism leading to cytopenias remains unresolved. To gain insight into this mechanism, bone marrow fibrosis (Fig. 1A) was found to occur in 88% of cases and is significantly associated with splenomegaly, the presence of cytopenias, and co- existence of autoimmune diseases. In this study, we are reporting the identification of the fibrosis initiating cell population. Methods Primary mesenchymal stromal cultures (MSCs) from LGL leukemia BM were isolated and grown under reduced oxygen conditions using undifferentiated and differentiation-specific culture conditions (Fig. 1B). These cultured cells were confirmed to deposit greater amounts of fibrillar Type I, III and V collagen matrix consistent with the heavy reticulin and trichrome positive stains observed in bone marrow biopsies (Fig. 1C). This collagen-producing cell population failed to support normal hematopoietic progenitor proliferation and colony formation in co-culture assays. Microarray analysis of the collagen-producing mesenchymal cell population was conducted and pathway analysis was performed using the GeneGo analysis platform from MetaCore™. Each list of genes was individually grouped and evaluated for membership on the set of GeneGo® pathway maps under the functional ontology enrichment tool. Calculation of p-values using hypergeometric distribution was used to determine pathway statistical significance. Flow cytometry was conducted to further evaluate the mesenchymal lineage. Results Supervised hierarchical clustering was performed to determine the overall similarity of LGL MSCs to publicly available, platform-compatible, microarrays from cells of mesenchymal lineage. Clustering showed that both patient and control MSCs display expression patterns distinct from committed mesenchymal lineages including osteoblasts, chondrocytes, and adipocytes. Mesenchymal-derived fibroblast differentiation can be distinguished by down-regulated CD29, CD44, CD105, CD106, CD117, bone morphogenetic protein receptor, and Sca-1 expression, and by up-regulated collagen type I, collagen type III, tenacin C, fibronectin, matrix metalloproteinase 1, fibroblast-specific protein 1, and vimentin. By microarray analysis and flow cytometry, we find no evidence that the cells are differentiated cells aside from collagen over expression. In order to define their pluripotent potential, sub-confluent cultures were grown in three types of differentiating media. Primary MSCs from both LGL leukemia patients and healthy controls were similar in their capacity to undergo trilineage differentiation into osteoblasts (stained with Alizarin Red S), adipocytes (Oil red O), and chondrocytes (Alican blue) (Fig. 1D). Next, pathway analyses revealed significant re-programming of the pluripotent MSCs. ECM production pathways were increased while proliferation pathways were down-regulated. Proliferation of MSCs is governed by production of autocrine growth factors. Both leukemia inhibitor factor (LIF) and basic fibroblast growth factor (FGFb) were significantly reduced compared to control MSCs (p<0.001) and the proliferative capacity of the LGL MSCs was severely diminished. Exogenous FGFb, but not LIF, restored LGLL MSC proliferation comparable to normal MSCs (Fig. 1E), normalized their spindle-shaped morphology, and maintained MSC plasticity. Interestingly, treatment with exogenous FGFb normalized collagen deposition (Fig. 1F) and restored hematopoietic supporting function. Conclusion STAT-activated LGL leukemia cells, like myeloproliferative neoplasms, share a propensity for the development of bone marrow fibrosis. These results indicate that MSCs from LGL leukemia are pluripotent, but are re-programmed to deposit excessive amounts of collagen. Restoration of FGFb maintains the pluripotency of these MSCs while reversing the fibrosis phenotype. Our data suggests that FGFb-cultured MSCs may be used to reverse cytopenias in LGL leukemia. Disclosures: No relevant conflicts of interest to declare.

Paroxysmal Nocturnal Hemoglobinuria: An Atypical Clinical Case Report and Proof of the Disease in Blood and BONE Marrow with FLOW Cytometry

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5166-5166
Author(s):  
Fabienne Pineau-Vincent ◽  
Pierre Lemaire ◽  
Habib Ghnaya ◽  
Guillaume Direz ◽  
Mohamed Kaabar ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Case Report ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Blood Smear ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Chromatin Condensation ◽  
Clinical Signs ◽  
Pnh Clone

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired disease, associated with hemolytic anemia and bone marrow failure. The cellular abnormality is a mutation in the phosphatidylinositol glycan class (PIG A) resulting in a deficiency of glycosylphosphadityl-inositol (GPI)-anchored complement regulatory proteins, including CD 55 and CD59, on the surface of blood cells. Case report We report the case of a French, 81 year-old-man, who was admitted to our institution with an unusual clinical presentation. He had a rheumatologic monitoring in the context of polyarthritis associated with anemia (98g/L). No hemolytic events were noticed and there was no notion of either transfusion. Biological results showed hemolytic regenerative anemia (98g/L) with 136G/L of reticulocytes, neutrophil polynuclears (4.2G/L) without degranulation and nevertheless rare degranulation cells, no blasts, normal level of platelets (258G/l), increase of LDH (Nx3), low haptoglobin (0.07g/L), negative direct Coombs test. The cytology aspect of medullar cells associated dysgranulopoiesis with degranulation of myeloid lineage and abnormal chromatin condensation, dyserythropoiesis, dysmegacaryopoiesis, in favor of a multilineage dysplasia without blasts. The marrow karyotype was normal. Due to the morphological results observed on the blood smear and their dissociation with the medullary cytology, flow cytometry (FC500) for GPI‘s expression study was performed. The used antisera were: CD55, CD59, CD14, CD16, CD24, CD66b, CD157, no FLEAR was tested. Results TableBloodBone marrowMononuclear cells CD14 FL378% intermediar cells70% negative cellsNeutrophil cells CD16 PE56% intermediar cells56% negative cellsNeutrophil cells CD66b FITC57% negative cells70% negative cellsGranular cells CD24 PE49% negative cells62% negative cellsRed cells CD55 FITC10% negative cells11% negative cellsRed cells CD59 FITC12% negative cells12% negative cells Figure 1 Blood Figure 1. Blood Figure 2 Bone Marrow Figure 2. Bone Marrow The confirmation was obtained by using CD157PE antisera on bone marrow with 70% negative mononuclear and granular cells. The results confirmed the PNH clone’s presence in the blood and also in bone marrow, and the results of flow cytometry could explain the cytological aspect of neutrophil polynuclear cells. It is rare to explore the expression of GPI molecules in bone marrow and there is no publication about the PNH clone whose identification required bone marrow cells for the confirmation of abnormalities in blood. Thus, the apoptosis in the bone marrow of the defective myeloid cells would explain the difference of granularity of polynuclear cells between bone marrow and blood smear. Conclusion The significance of this observation is related to the search of a PNH clone when cytological dissociation is observed between the peripheral blood and bone marrow, associated with biological hemolysis arguments (increased LDH and decreased haptoglobin). It is well known that 6 at 8% of myelodysplasia had PNH clone; the originality of this case report is the initial clinical signs and the laboratory proof of PNH in the blood and the bone marrow. This observation was submitted at the national reference center of PNH in France (St Louis Hospital - Hematology Department - Professor SOCIE) and the treatment by eculizumab was introduced. Disclosures No relevant conflicts of interest to declare.

scholarly journals Daratumumab Interferes with Flow Cytometric Evaluation of Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5630-5630 ◽  
Author(s):  
Sudhir Perincheri ◽  
Richard Torres ◽  
Christopher A Tormey ◽  
Brian R Smith ◽  
Henry M Rinder ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Population ◽  
Plasma Cell ◽  
Light Chain ◽  
Plasma Cells ◽  
Kappa Light Chain ◽  
Positive Events ◽  
History Of

Abstract The diagnosis of multiple myeloma (MM) requires the demonstration of clonal plasma cells at ≥10% marrow cellularity or a biopsy-proven bony or extra-medullary plasmacytoma, plus one or more myeloma-defining events. Clinical laboratories use multi-parameter flow cytometry (MFC) evaluation of cytoplasmic light chain expression in CD38-bright, CD45-dim or CD138-positive, CD45dim cells to establish plasma cell clonality with a high-degree of sensitivity and specificity. Daratumumab, a humanized IgG1 kappa monoclonal antibody targeting CD38, has been shown to significantly improve outcomes in refractory MM, and daratumumab was granted breakthrough status in 2013. Daratumumab is currently approved for treatment of MM patients who have failed first-line therapies. It has been noted that daratumumab can interfere in blood bank assays for antibody screening, as well as serum protein electrophoresis (SPEP). We describe for the first time daratumumab interference in the assessment of plasma cell neoplasms by MFC; daratumumab interfered with both CD38- and CD138-based gating strategies in three MM patients. Patient A is a 68 year old man with a 10 year history of MM who had failed multiple therapies. He had then been treated with daratumumab for two months, stopping therapy 25 days prior to bone marrow assessment. Patient B is a 53 year old man with a 3 year history MM who had failed numerous treatments. He had been receiving daratumumab monotherapy for two months at the time of his bone marrow studies. On multiple marrow aspirates at times of relapse prior to receiving daratumumab, both patients had demonstrated CD38-bright positive CD45dim/negative plasma cells expressing aberrant CD56, as well as kappa light chain restriction; mature B cells were polyclonal in both. Patient C is a 65 year old man with a four-year history of MM status post autologous stem cell transplantation, who had been receiving carfilzomib and pomalidomide following relapse and continues to have rising lambda light chains and rib pain. He now has abnormal plasma cells in blood worrisome for plasma cell leukemia. Bone marrow aspirates from patients A and B, and blood from patient C demonstrated near absence of CD38-bright events as detected by MFC (Figure 1). Hypothesizing that these results were due to blocking of the CD38 antigen by daratumumab, gating on CD138-positive events was assessed; surprisingly, virtually no CD138-positive events were detected by MFC. All 3 samples demonstrated a CD56-positive CD45dim population; when light chain studies were employed using specific gating on the CD56-positive population, light chain restriction was demonstrated in all patients (Figure 1). Aspirate morphology confirmed numerous abnormal, nucleolated plasma cells (Figure 2A), thus excluding a sampling error. CD138 and CD38 expression was also tested on the marrow biopsy cores from both patients. In contrast to MFC, immunohistochemistry (IHC) showed positive labeling of plasma cells with both CD138 (Figure 2B) and CD38 (Figure 2C). The reason for the labeling discrepancy between MFC and IHC is unknown. The different antibodies in the assays may target different epitopes; alternatively, tissue fixation/decalcification may dissociate the anti-CD38 therapeutic monoclonal from its target. Detection of clonal plasma cell populations is important for assessing response to therapy. Laboratories relying primarily on MFC to assess marrow aspirates without a concomitant biopsy may falsely diagnose remission or significant disease amelioration in daratumumab-treated patients. MFC is generally highly sensitive for monitoring minimal residual disease (MRD) in MM, but daratumumab-treated patients should have their biopsy evaluated to confirm the MRD assessment by MFC. We were able to detect large numbers of plasma cells and also demonstrate clonality in our patients based on an alternative MFC marker, aberrant CD56 expression, an approach that may not be possible in all cases. Figure 1 Flow cytometry showing near-absence of CD38-bright elements in the marrow of patient A (top panels). Gating on CD56-positive cells in the same sample reveals a kappa light chain-restricted plasma cell population (bottom panels). Figure 1. Flow cytometry showing near-absence of CD38-bright elements in the marrow of patient A (top panels). Gating on CD56-positive cells in the same sample reveals a kappa light chain-restricted plasma cell population (bottom panels). Figure 1 The marrow aspirate from Fig. 1 shows abnormal plasma cells (A). Immunohistochemistry on the concomitant biopsy shows the presence of numerous CD138-positive (B) and CD38-positive (C) plasma cells. Figure 1. The marrow aspirate from Fig. 1 shows abnormal plasma cells (A). Immunohistochemistry on the concomitant biopsy shows the presence of numerous CD138-positive (B) and CD38-positive (C) plasma cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals MELPHALAN-INDUCED CYTOTOXICITY IN THE BONE MARROW OF RATS BY FLOW CYTOMETRY MEASUREMENTS

2019 ◽  
Vol 4 (2) ◽  
pp. 72-78
Author(s):  
B I. Gerashchenko ◽  
I. M. Todor ◽  
O. O. Shevchuk ◽  
V. G. Nikolaev
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acridine Orange ◽  
Cell Population ◽  
Drug Administration ◽  
Cytotoxic Effect ◽  
Cytotoxic Agents ◽  
Experimental Conditions ◽  
Chemotherapy Drugs ◽  
Polychromatic Erythrocytes

Background. Bone marrow (BM) that contains hematopoietic cells of various lineages is a sensitive target for a number of cytotoxic agents including chemotherapy drugs. Objective. Flow cytometry (FCM) was chosen to test cytotoxicity in BM of rats, that received melphalan either intravenously (i.v.) or intraperitoneally (i.p.). Methods. One group of rats received melphalan i.v. (3 mg/kg) followed by the BM examination on the 3rd and 7th day after drug administration, whereas another group of animals received this drug i.p. in total doses of 9 and 15 mg/kg followed by the BM examination on the next day after the 3rd and 5th injection of the drug. BM cells were stained with acridine orange and analyzed by FCM. Cytotoxicity was assessed by determining the percentage of total nucleated cells (TNC%) among the whole BM cell population and by determining the percentage of polychromatic erythrocytes (PCE%) among the whole population of enucleated erythrocytes. Results. Regardless of the dose and regimen of melphalan administration, either i.v. or i.p. administered drug caused a significant reduction of TNC%. On the average, the i.p. administered drug resulted in about 2.0-fold decrease of TNC% (P<0.05), while the i.v. administered drug resulted in about 1.3-fold decrease of TNC% (P<0.05). As for enucleated erythrocytes, the i.p. administered drug resulted in about 1.4-fold decrease of PCE% (P<0.05), whereas the i.v. administered drug did not cause any changes in the PCE%. Conclusions. Under these experimental conditions, i.p. administrated melphalan is considerably more cytotoxic than i.v. administered melphalan. This cytotoxic effect is preferentially due to impaired erythropoiesis.

Multiparameter Flow Cytometry For Detection Of Minimal Residual Disease In Multiple Myeloma After T-Cell Depleted Allogeneic Stem Cell Transplant

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4647-4647
Author(s):  
Satyajit Kosuri ◽  
Katherine M Smith ◽  
Deborah Kuk ◽  
Sean M. Devlin ◽  
Peter G. Maslak ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Cell Population ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Residual Disease ◽  
Multiparameter Flow Cytometry

Introduction Multiparameter flow cytometry (MFC) has been shown to be a sensitive, reproducible and broadly applicable method for the early detection of minimal residual disease (MRD) in the bone marrow (BM) of pts with multiple myeloma (MM) following induction chemotherapy and/or autologous stem cell transplantation. In this study, we were interested in assessing the potential of MFC as a reliable and potentially predictive marker in pts with multiple myeloma who have undergone T-cell depleted allogeneic hematopoietic stem cell transplantation (TCD HSCT). Methods We analyzed the results of MFC obtained in 35pts with multiply relapsed MM, who also have high-risk cytogenetics undergoing allo TCD-HSCT from HLA compatible related (n= 15) and unrelated (matched (n=8), mismatched (n=12) donors. We compared these results to standard myeloma markers obtained from the blood and marrow of these pts at days 30, 60-90, 120-180, 12 and 24 months routinely and as clinically indicated thereafter post TCD HSCT. Disease evaluation included serologic immunoglobulin levels, serum protein electrophoresis/immunofixation, and serum analysis of free light chains, bone marrow biopsy and aspirate. Bone marrow specimens from each time point were also analyzed by MFC with a panel including CD38, CD56, CD45, CD19, CD138, cyKAPPA, and cyLAMBDA by gating on distinct populations of bright CD38+/CD45- plasma cells at 200,000 acquired events total or at least 100 gated plasma cell events. Malignant plasma cells (MPC) were defined as CD38+/CD138+/CD56+/CD45- and/or positive for light chain clonal excess. MPC were detected in the BM sample at the MFC sensitivity of 10-4(>1 MPC in 104normal cells). Results Thirty-five pts with multiply relapsed MM undergoing allo TCD HSCT were analyzed over median follow up of 27 months (range 6.2 – 53.3). Eighteen/35 pts did not relapse during the follow up period and none of these pts had a detectable CD38+/CD138+/CD56+/CD45- cell population by MFC. Seventeen/35 pts developed relapsed disease at a median of 12.5 months (range 3.2 – 52.5) post allo TCD-HSCT by standard serologic markers and all pts were found to be positive by MFC. The percentages of bright CD38+/CD45- cells in these pts ranged from 0.01% to 16.05% at time of first detection. In 14/17 pts, MFC became positive concurrently with standard serologic myeloma markers at relapse. In 3/17 pts, MFC detected a malignant plasma cell population with aberrant phenotype of 0.068%, 0.043% and 0.012% at 48.2, 24 and 25.4 months, respectively, post TCD HSCT in the absence of other positive markers in blood and bone marrow. These pts were also immunofixation (IF) negative at conversion to MFC positivity. Subsequent follow up of studies of these 3 pts lead to detection of recurrence by IF and/or M-spike/ aspirate at 3.8, 1.8 and 8.7 months with median follow up of 150 days after first MFC detection. The populations of MPC initially detected by MFC had increased upon relapse to higher levels. Interestingly, in 2 pts we detected 6 and 8% plasma cells by bone marrow aspirate at 90 days and 180 days, respectively, post TCD HSCT, while flow cytometry detected only CD138+/CD56-/CD45+ cells. These 2 pts never relapsed and continued to remain in CR without further intervention. Conclusions These analyses demonstrate that MFC performed on marrow specimen of pts with relapsed MM who underwent a TCD HSCT provides additional important results to assess the overall disease status. A negative MFC indicated non relapse 100% of the time attesting to its negative predictive value. In all of our patients diagnosed with relapsed MM by traditional parameters, MFC was concurrently positive. Importantly, in 3/17 pts (18%) MRD detected MPC prior to overt relapse. Interestingly, MFC was able to detect false positive marrow relapses as well. Therefore, MFC permits the detection of MRD preceding frank relapse and can distinguish a malignant plasma cell population from proliferating recovering marrow post transplant. In the post allo TCD-HSCT setting MFC may serve as an early marker which can help formulate the timing of therapeutic interventions, such as adoptive immunotherapeutic approaches, as MFC detection provides a window of several weeks to initiate treatment before disease recurrence by serology. Disclosures: No relevant conflicts of interest to declare.

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