The Expanding Diagnostic Role of Flow Cytometry in Bone Marrow Studies of Patients with Lymphomas and Plasma Cell Disorders

2012 ◽  
pp. 47-65
Author(s):  
Anna Porwit
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Plasma Cell ◽  
Plasma Cell Disorders ◽  
Diagnostic Role

The role of bone marrow biopsy in patients with plasma cell disorders; should all patients with a monoclonal protein be biopsied?

2019 ◽  
Vol 19 (10) ◽  
pp. e333
Author(s):  
M Hasib Sidiqi ◽  
Mohammed Aljama ◽  
Shaji Kumar ◽  
Dragan Jevremovic ◽  
Francis Buadi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Biopsy ◽  
Plasma Cell ◽  
Monoclonal Protein ◽  
Plasma Cell Disorders

scholarly journals The role of bone marrow biopsy in patients with plasma cell disorders: should all patients with a monoclonal protein be biopsied?

2020 ◽  
Vol 10 (5) ◽  
Author(s):  
M. Hasib Sidiqi ◽  
Mohammed Aljama ◽  
Shaji K. Kumar ◽  
Dragan Jevremovic ◽  
Francis K. Buadi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Biopsy ◽  
Plasma Cell ◽  
Monoclonal Protein ◽  
Plasma Cell Disorders

scholarly journals Biological properties of bone marrow plasma cells influence their recovery in aspirate specimens: impact on classification of plasma cell disorders and potential bias to evaluation of treatment response

2020 ◽  
Vol 99 (11) ◽  
pp. 2599-2609
Author(s):  
Svitlana Demyanets ◽  
Alexandra Kaider ◽  
Ingrid Simonitsch-Klupp ◽  
Günther Bayer ◽  
Almira Subasic ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Cell Phenotype ◽  
Special Focus ◽  
Plasma Cell Disorders ◽  
Bone Marrow Plasma ◽  
The Impact

Abstract Methods to estimate bone marrow plasma cells (BMPC) basically include histopathology, cytomorphology, and flow cytometry. The present study compares the outcomes of these methods with special focus on the impact of BMPC-specific characteristics on their recovery by either method. Laboratory reports of diagnostic samples from 238 consecutive patients with suspected or known plasma cell disease were retrospectively analyzed. The median (IQR) proportion of BMPC was 30.0% (15.0–70.0%) by histological review (hBMPC), 7.0% (2.0–16.0%) by smear review (sBMPC), and 3.0% (0.8–10.0%) by flow cytometry (fBMPC). The disparity of results between core biopsy and aspirate smear was enhanced in case of poor quality of the smear, increased BM fiber content, higher grade cell atypia, expression of CD56 (all P < 0.0001), the number of cytogenetic aberrations (P = 0.0002), and abnormalities of the MYC gene (P = 0.0002). Conversely, expression of CD19 and a non-clonal plasma cell phenotype were associated with a lower difference between hBMPC and sBMPC (both P < 0.0001). The disparity between the percentages of sBMPC and fBMPC was associated with the quality of the smear (P = 0.0007) and expression of CD56 (P < 0.0001). Our results suggest that the recovery of BMPC in aspirate specimens not only is a matter of sampling quality but also depends on biological cell properties. Aspiration failure due to malignant type features of BMPC may lead to misclassification of plasma cell disorders and represent a bias for the detection of minimal residual disease after therapy.

scholarly journals Levels of CEACAM6 in Peripheral Blood Are Elevated in Patients with Plasma Cell Disorders: A Potential New Diagnostic Marker and a New Therapeutic Target?

2019 ◽  
Vol 2019 ◽  
pp. 1-6
Author(s):  
N. Steiner ◽  
R. Hajek ◽  
D. Nachbaur ◽  
B. Borjan ◽  
S. Sevcikova ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Plasma Cell ◽  
Adhesion Molecule ◽  
Peripheral Blood ◽  
Therapeutic Target ◽  
Plasma Levels ◽  
Diagnostic Marker ◽  
Healthy Controls ◽  
Plasma Cell Disorders

Introduction. The prognosis of multiple myeloma is still unfavorable due to inherent characteristics of the disease and the often-delayed diagnosis due to widespread and unspecific symptoms such as back pain and fatigue. Therefore, a simple diagnostic blood test would be helpful to speed up the diagnostic procedure in such patients (pts.). Here, we evaluated the diagnostic value of plasma levels of carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) in the peripheral blood and bone marrow of pts. with plasma cell disorders and in healthy controls. Materials and Methods. Immunoreactive CEACAM6 was determined in the peripheral blood and bone marrow (n=95/100) of pts. with monoclonal gammopathy of unknown significance (MGUS: 28/37), newly diagnosed multiple myeloma (NDMM: 42/40), and relapsed/refractory multiple myeloma (RRMM: 25/23) by sandwich ELISA. Results. Median CEACAM6 levels in the peripheral blood of pts. with plasma cell disorders were significantly higher than those of healthy controls (healthy controls: 15.2 pg/ml (12.1-17.1); MGUS: 19.0 pg/ml (16.4-22.5); NDMM: 18.0 pg/ml (13.4-21.2); and RRMM: 18.9 pg/ml (15.2-21.5); p<0.001). Plasma levels of CEACAM6 discriminated healthy subjects from MGUS/NDMM pts. (AUC=0.71, 95% CI: 0.6-0.8); i.e., a CEACAM6 level>17.3 pg/ml has an 82% (95% CI: 70-90) predictive probability for the identification of MGUS or NDMM. Moreover, CEACAM6 levels in the bone marrow were significantly higher in RRMM pts. than in NDMM pts. (p=0.04), suggesting a role of this molecule in disease progression. Conclusion. CEACAM6 plasma levels can noninvasively identify pts. with a plasma cell disorder and should be evaluated prospectively as a potential diagnostic marker. Moreover, due to high CEACAM6 levels in the bone marrow in RRMM pts., this adhesion molecule might be a therapeutic target in multiple myeloma pts.

Abstract 655: Hyperdiploidy in plasma cell disorders using multi-parametric flow cytometry (MFC) vs. FISH

2018 ◽  
Author(s):  
Surbhi Sidana ◽  
Nidhi Tandon ◽  
Dragan Jevremovic ◽  
Rhett P. Ketterling ◽  
Angela Dispenzieri ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Plasma Cell ◽  
Plasma Cell Disorders

Incomplete Presentation of WHIM Syndrome: The Diagnostic Role of Dysmorphic Neutrophils in Bone Marrow

2020 ◽  
Vol 42 (7) ◽  
pp. 449-450
Author(s):  
Kousaku Matsubara ◽  
Aya Iwata ◽  
Yu Kawasaki ◽  
Yoshitaka Honda ◽  
Takahiro Yasumi
Keyword(s):  
Bone Marrow ◽  
Whim Syndrome ◽  
Diagnostic Role

Role of Il-2, Il-7, and IL-15 in T Cell Mediated Graft-vs-Leukemia Against Human Acute Lymphoblastic Leukemia in a NOD/scid Chimeric Mouse Model.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5256-5256
Author(s):  
Doug Cipkala ◽  
Kelly McQuown ◽  
Lindsay Hendey ◽  
Michael Boyer
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Mouse Model ◽  
Scid Mouse ◽  
In Vitro Cytotoxicity ◽  
Scid Mouse Model

Abstract The use of cytotoxic T-lymphocytes (CTL) has been attempted experimentally with various tumors to achieve disease control. Factors that may influence GVT include CTL cytotoxicity, ability to home to disease sites, and survival of T cells in the host. The objective of our study is to evaluate the GVL effects of human alloreactive CTL against ALL in a chimeric NOD/scid mouse model. CTL were generated from random blood donor PBMCs stimulated with the 697 human ALL cell line and supplemented with IL-2, -7, or -15. CTL were analyzed for in vitro cytotoxicity against 697 cells, phenotype, and in vitro migration on day 14. NOD/scid mice were injected with 107 697 ALL cells followed by 5x106 CTL. Mice were sacrificed seven days following CTL injection and residual leukemia was measured in the bone marrow and spleen via flow cytometry. The ratios of CD8/CD4 positive T cells at the time of injection were 46/21% for IL-2, 52/31% for IL-7, and 45/14% for IL-15 cultured CTL (n=13). Control mice not receiving CTL had a baseline leukemia burden of 2.01% and 0.15% in the bone marrow and spleen, respectively (n=15). Mice treated with IL-15 cultured CTL had a reduction in tumor burden to 0.2% (n=13, p=0.01) and 0.05% (n=13, p=0.01) in bone marrow and spleen, respectively. Those treated with IL-2 or IL-7 cultured CTL showed no significant difference in leukemia burden in either the bone marrow (IL-2 1.28%, Il-7 5.97%) or spleen (IL-2 0.4%, IL-7 0.33%). No residual CTL could be identified in the bone marrow or spleen at the time of sacrifice in any CTL group. CTL grown in each cytokine resulted in similar in vitro cytotoxicity at an effector:target ratio of 10:1 (IL-2 41.3%, IL-7 37.7%, IL-15 45.3%, n=12–15, p&gt;0.05 for all groups) and had statistically similar intracellular perforin and granzyme-B expression. In vitro CTL migration to a human mesenchymal stem cell line was greatest with IL-15 CTL (30.5%, n=4), followed by IL-7 CTL (18.9%, n=4), and least in IL-2 CTL (17.9%, n=4), though the differences were not significant. In vitro CTL migration was analyzed to an SDF-1α gradient as CXCR4/SDF-1α interactions are necessary for hematopoietic progenitor cell homing to the bone marrow. IL-15 cultured CTL showed the highest migration (48.8%, n=8) as compared to IL-2 (21.7%, n=6, p=0.048) or IL-7 CTL (35.9%, n=8, p&gt;0.05). However, surface expression of CXCR4 measured by flow cytometry was significantly higher in IL-7 CTL (89.4%, n=9) compared to IL-2 CTL (52.2%, n=9, p&lt;0.001) and IL-15 CTL (65.4%, n=10, p=0.002). Experiments are currently underway to further evaluate the role of CXCR4/SDF-1α in GVL. Preliminary in vivo experiments do not suggest any significant differences in CTL engraftment when evaluated at 24 hours post injection. Expression of the anti-apoptotic bcl-2 protein was greatest on IL-7 (MFI=5295, n=13) and IL-15 (MFI=4865, n=14) when compared to IL-2 CTL (MFI=3530, n=13, p=0.02 vs. IL-7, p=0.05 vs. IL-15), suggesting an increased in vivo survival ability. We hypothesize that IL-15 cultured CTL have greater GVL effects due to either higher in vivo survival, greater bone marrow homing efficiency, or both. Future experiments are planned to evaluate in vivo administration of IL-2 to enhance CTL survival in the host. In conclusion, IL-15 cultured CTL had significantly greater in vivo GVL effects compared to IL-2 and IL-7 CTL in the NOD/scid mouse model. This model can be utilized to evaluate the mechanism of T cell mediated GVL against ALL and potentially other human malignancies.

Application of Multiparameter Flow Cytometry for the Identification of Dysplasia in Granulocytic, Monocytic, and Erythrocytic Lineages and Blasts in Patients with Myelodysplastic Syndromes.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2615-2615
Author(s):  
Wolfgang Kern ◽  
Claudia Schoch ◽  
Susanne Schnittger ◽  
Torsten Haferlach
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Myelodysplastic Syndromes ◽  
Erythroid Cell ◽  
Multiparameter Flow Cytometry ◽  
Cell Populations ◽  
Aberrant Expression ◽  
Hla Dr ◽  
Cd16 Expression

The diagnosis and classification of myelodysplastic syndromes (MDS) are based on cytomorphology (CM) and cytogenetics. A high degree of experience in CM is required to allow the accurate identification of dysmyelopoiesis and quantification of bone marrow blasts. The identification of dysplastic features in all lineages by multiparameter flow cytometry (MFC) has been shown feasible. To further analyze the potential role of MFC in the diagnostic work-up of MDS we analyzed 224 bone marrow samples from patients with suspected of proven MDS by MFC, CM, and cytogenetics in parallel. Blast counts as determined by CM and MFC, respectively, ranged from 0% to 21% (median, 5%) and from 0% to 33% (median, 4%; correlation: r=0.192, p=0.018). The median number of aberrant features detected by MFC were 0 for blasts (range, 0 to 4), 2 for granulocytes (0 to 7), 1 for monocytes (0 to 5), and 0 for erythrocytes (0 to 2). The most frequent dysplastic features observed in the blast populations included aberrant coexpression of CD11b (20.5%), CD15 (14.3%) and CD64 (14.3%). The most frequent dysplastic features observed in the granulocytic cell populations included reduced side-scatter signal corresponding to hypogranulation (71.4%), aberrant coexpression of CD56 (29.0%), aberrant pattern of CD13/CD16 expression (26.3%), aberrant pattern of CD11b/CD16 expression (25.9%), reduced expression of CD64 (17.0%), and aberrant expression of HLA-DR (14.7%). The most frequent dysplastic features observed in the monocytic cell populations included aberrant coexpression of CD56 (31.3%), aberrant coexpression of CD16 (26.3%), an aberrant pattern of CD11b/HLA-DR expression (6.7%), and aberrant coexpression of CD2 (5.8%). The most frequent dysplastic features observed in the erythroid cell populations included an aberrantly strong expression of CD71 and CD235a (23.7%), a lack of CD71 expression (10.7%), and an aberratly homogeneous expression of CD71 (7.1%). The presence of dysplastic features by CM as well as the presence of cytogenetic aberrations tended to be associated with a higher number of dysplastic features by MFC. These data suggest that the identification of dysplastic features by MFC is feasible although there is a large heterogeneity in aberrantly expressed antigens. Thus, a comprehensive panel of antibodies must be applied to allow the detection of dysplasia. Future studies will define the role of MFC in optimizing the diagnosis of MDS in cooperation with CM and cytogenetics.

Deregulation of TNFRSF18 (GITR) Through Promoter CpG Island Methylation Induces Tumor Proliferation in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2424-2424
Author(s):  
Yang Liu ◽  
Yong Zhang ◽  
Phong Quang ◽  
Hai T Ngo ◽  
Feda Azab ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Cpg Island ◽  
Advisory Committees ◽  
Brdu Assay

Abstract Abstract 2424 Introduction Tumor necrosis factor receptor super families (TNFRSFs) play an important role in activation of lymphocyte and cell apoptosis. However the function of TNFRSFs in multiple myeloma (MM) remains unknown. Loss of function mutation of Fas antigen (TNFRSF6) was identified in MM cells, thus suggesting the possible role of TNFRSFs in regulating MM pathogenesis. We therefore investigated the epigenetic mechanisms that may mediate inactivation of TNFRSFs and its functional role in MM. Methods Dchip software was utilized for analyzing gene expression dataset. DNA was extracted from both primary CD138+ MM plasma cells and MM cell lines using blood & tissue DNA isolation kit (Qiagen, Inc.). Expression of GITR in primary CD138+ plasma cells was detected by Imunohistochemistry (IHC) DNA methylation was analyzed by methylated DNA immunoprecipitation (Medip) assay and bisulfate sequencing. 5'azacytidine was used to demethylate genomic DNA. Gene expression was detected by qRT-PCR and confirmed at the protein level by flow cytometry and western-blot. Over-expression of GITR was obtained in MM1.S cells by using GITR recombinant plasmid and electroporation. Apoptosis was determined using Annexin/PI staining and flow cytometry analysis. Activation of apoptotic signaling was studied by western blot. Cell survival and proliferation were analyzed by MTT and BrdU assay, respectively. Recombinant GITR-lentivirus was obtained from the supernatant of culture medium after 72 hours transfection in 293 cells. GFP positive MM cells were sorted and analyzed by flow cytometry. In vivo effect of GITR on MM tumor growth was determined by injection of GITR over-expressing MM cells in null mice. Mice skull, femur and vertebrae were isolated after 4 weeks injection. Anti-human CD138+ mAb microbead was used to detect MM cells extracted from mice tissue by flow cytometry. Results Gene-expression profiling showed down-regulation of TNFRSFs, including TNFRSF11A, TNFRSF11B, TNFRSF8, TNFRSF10C, TNFRSF9, TNFRSF21, TNFRSF1B, TNFRSF1A and TNFRSF18, compared to normal plasma cells. Moreover, Our IHC results also showed that GITR expression was positive in primary CD138+ plasma cells from 9 normal bone marrow, but negative in 9 MM samples. Importantly, we found that low GITR expression significantly correlated with MM progression. Indeed, GITR gene levels were lower in smoldering and active MM patients compared to MGUS patients and normal donors. Promoter CpG island (CGI) methylation of GITR was indentified in 5 out of 7 MM primary bone marrow (BM)-derived CD138+ cells but not in normal BM-derived plasma cells. Bisulfate sequencing and Medip assay showed that methylation of GITR was significantly associated with GITR expression in 5 MM cell lines, including MM1.S, OPM1, U266, RPMI and INA6. Promoter CGI of GITR was highly methylated leading to complete silencing of GITR in MM1.S cell line. GITR expression was significantly up-regulated in MM cells upon treatment with the 5'azacytidine. MTT and BrdU assay revealed that the proliferation and survival of MM1.S cells was disrupted in the GITR over-expressing MM1.S cells, notably with inhibition of cell proliferation compared to control vector infected cells. Moreover induction of cytotoxicity in GITR over-expressing cells was confirmed by using GFP competition assay. GITR-induced apoptosis was supported by induction of caspase 8 and 3 cleavage. The inhibition of human CD138+ plasma cell growth in the bone marrow of SCID mice using a disseminated MM xenograft model was observed in the experimental group injected with GITR expressing cells compared to the control group after 4 weeks injection. Conclusion Our findings uncovered a novel epigenetic mechanism contributing to MM pathogenesis, showing the role of GITR methylation as a key regulator of MM cell survival. Disclosures: Roccaro: Roche:. Ghobrial:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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