Quantitation of Plasma Cells in Bone Marrow Aspirates by Flow Cytometric Analysis Compared With Morphologic Assessment

2007 ◽  
Vol 131 (6) ◽  
pp. 951-955
Author(s):  
Kristi J. Smock ◽  
Sherrie L. Perkins ◽  
David W. Bahler
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Plasma Cell ◽  
Gold Standard ◽  
Plasma Cells ◽  
Flow Cytometric Analysis ◽  
Flow Cytometric ◽  
Bone Marrow Plasma ◽  
Plasma Cell Dyscrasias ◽  
Cellular Processing

Abstract Context.—Accurate quantitation of bone marrow plasma cells is an important component in the diagnosis and posttreatment assessment of plasma cell dyscrasias. Although flow cytometry is sometimes used for this purpose and can rapidly evaluate many cells, the accuracy of flow-based plasma cell quantitation compared with morphologic assessment (currently the gold standard) is uncertain as direct comparison studies have not been previously reported. Objective.—To determine how percentages of plasma cells in diagnostic aspirate smears quantitated by morphologic assessment relate to percentages of plasma cells quantified by flow cytometry. Design.—Thirty bone marrow cases with 10% or more plasma cells and leukemia/lymphoma flow cytometry immunophenotyping studies were identified from our hematopathology database. The Wright-stained aspirate smears, marrow biopsy sections, and flow cytometry histograms were reviewed. Results.—Morphologically determined plasma cell percentages from the diagnostic aspirate smears were consistently higher than those determined by flow cytometry. Much of this difference appeared to be related to differences in sample quality. However, the cellular processing involved in performing flow cytometry also appeared to reduce plasma cell percentages in many cases. Conclusions.—This study helps define the limitations of flow cytometry for quantitating plasma cell loads in marrow aspirate specimens that may significantly affect the diagnosis or assessment of treatment response.

CD 138 Immunostaining of Bone Marrow Trephine Specimens Is the Most Sensitive Method for Quantifying Marrow Involvement in Patients with Plasma Cell Dyscrasias.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5071-5071
Author(s):  
Ashley P. Ng ◽  
Andrew Wei ◽  
Dinesh Bhurani ◽  
Peter Chappell ◽  
Frank Feleppa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Flow Cytometric Analysis ◽  
T Test ◽  
P Value ◽  
Flow Cytometric ◽  
Paired T Test ◽  
Sensitive Method

Abstract AIM In this study plasma cell quantitation comparing four modalities comprising morphology of the aspirate, flow cytometry, HEtrephine section examination and bone marrow immunohistology was undertaken to determine the most sensitive technique. METHODS 70 patients with a plasma-cell dyscrasia were retrospectively analysed. Plasma-cell quantitation was performed on Romanowsky-stained aspirate slides by a 200-cell differential. Flow cytometric quantitation of plasma-cell burden was performed by identifying CD38/138 co-expressing cells. Trephine specimens were analysed after HEand CD138 staining. Statistical analysis was performed using a two-tailed paired t-test. RESULTS See Table 1. DISCUSSION Significant discrepancy was noted between the four methods for determining plasma-cell infiltration. CD138 immunohistochemical staining of paraffin embedded trephine specimens was the most sensitive modality. Flow-cytometric analysis underestimated the degree of marrow infiltration. Morphology of the aspirate sample may also underestimate plasma-cell burden as sampling may be affected by patchy marrow infiltration as well hypoplastic, fibrotic marrows or clotted specimens. Quantification of plasma cells based on HEstained trephine specimens is less sensitive, and is dependent on the technical quality of the specimen and observer experience. Immunostaining has the advantages of improved plasma-cell identification compared to HEsections and avoids the problems inherent in the analysis of aspirate samples. In conclusion, CD138 immunostaining is the most sensitive method for quantifying plasma-cell burden in the bone marrow. Two tailed Paired t-test analysis of Quantitation of Plasma Cell burden by various modalities Flow Cytometry Aspirate Hematoxylin & Eosin Immunostaining Mean % of plasma cells 5.6 18.9 23.6 29.7 Paired t-test (two tailed) Mean Difference compared to CD138 immunostaining −24.12 −11 −6.14 - 95% C.I. −30.5 to −17.8 −15.8 to −6.1 −8.34 to −3.9 - p-value p<0.0001 p<0.0001 p<0.0001 -

Evaluation of a 5-Color Flow Cytometric Technique for Immunophenotyping and Quantitation of Plasma Cell Myeloma in Post-Therapy Bone Marrows.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5036-5036
Author(s):  
Tove Isaacson ◽  
Andrzej Jakubowiak ◽  
Lloyd Stoolman ◽  
Usha Kota ◽  
William Finn ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Minimal Residual Disease ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Residual Disease ◽  
Plasma Cell Myeloma ◽  
Color Flow ◽  
Flow Cytometric ◽  
Minimal Residual

Abstract Multiparameter flow cytometry is a useful tool for comprehensive immunophenotyping of plasma cell myeloma, and has been proposed as a sensitive method for the evaluation of minimal residual disease in patients following treatment. This study aimed to assess the value of flow cytometry in quantitation of residual disease, in comparison to routine morphologic examination of first-pull bone marrow aspirate smears, in myeloma patients post-therapy. Heparinized bone marrow aspirates were obtained from 27 treated patients with plasma cell myeloma. Cells were prepared for 5-color flow cytometric analysis within 24-hours of specimen draw. Surface membrane staining with anti-CD19, CD20, CD38, CD45, CD56, and CD138 was followed by ammonium chloride lysis of red cells. Fixed and permeabilized cells were analyzed for cytoplasmic light chains to confirm clonality. Data were acquired using an FC500 flow cytometer (Beckman-Coulter), analyzed with CXP software with plasma cells isolated based on bright CD38+ or CD138+ expression. A median of 97,639 cellular events (range 14,279 to 262,508) were collected per analysis. Flow cytometric enumeration of plasma cells was compared to 500-cell differential counts of Wright-Giemsa-stained first-pull aspirate smears from the same cases. The median plasma cell count as determined by flow cytometry was 0.5% (range 0–7.9%). The median plasma cell count estimated by morphologic review was 8.0% (range 0–84.4%). Flow cytometry underestimated the plasma cell content in all but one case. Clonal plasma cells expressed CD38 and CD138 in all cases; 87.5% (21/24) coexpressed CD56, 25% (6/24) coexpressed CD45, and 4.2% (1/24) coexpressed CD19. None was positive for CD20. Although detection of minimal residual disease after therapy for acute leukemia is routinely achieved by flow cytometric analysis, successful quantitation of minimal residual disease in treated myeloma patients using flow cytometry remains limited as it usually underestimates the plasma cell content of bone marrow samples compared to routine morphology of first-pull aspirates. We have observed that this holds true for both pre-treatment and post-treatment specimens. Causes for the discrepancy may include hemodilution of second-pull aspirates used for flow cytometry, fragility and loss of plasma cells during preparation for flow cytometry, and incomplete disaggregation of plasma cells from bone marrow spicules. With improved outcome of treatments, better and more reliable methods of detection of minimal residual disease are needed for optimal prognostic stratification. We are currently validating alternative methods, which may offer more sensitivity while at the same time allow more objectivity, for assessing the amount of minimal residual disease in myeloma patients.

Humoral Immunity and Plasma Cell Changes in Patients Responding to CD19-Specific Chimeric Antigen Receptor (CAR)-Modified T-Cell Adoptive Immunotherapy

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1110-1110
Author(s):  
Vijay Bhoj ◽  
Michael C Milone ◽  
Carl H. June ◽  
David Porter ◽  
Stephan A. Grupp ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Humoral Immunity ◽  
Research Funding ◽  
Plasma Cells ◽  
Flow Cytometric ◽  
Antibody Titers

Abstract Introduction: T cells engineered to express chimeric antigen receptors (CARs) recognizing CD19 (CART19) can eliminate malignant cells in acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL). We and other groups have shown that persistent tumor eradication by CD19-specific T cell immunotherapy is accompanied by normal B-cell aplasia. It is assumed that responding patients cannot make new antibody responses post-successful CART19 treatment; however, the status of previously established humoral immunity in these patients is currently unknown. Understanding the consequence of successful CART19 therapy on established humoral immunity has implications for both the clinical management of CART19-treated patients as well as the potential application of this therapy to non-malignant diseases such as autoimmunity and transplantation. Methods: We performed a prospective, observational study of adult and pediatric patients with ALL and adults with relapsed/refractory CLL, who were enrolled in clinical trials of CART19 at our institution. Serum antibody titers to previously-generated vaccine or vaccine-related pathogens (Streptococcus pneumoniae, Tetanus toxoid, Hemophilus influenza type-B (HIB), Measles, Mumps, and Rubella) were determined along with a quantitative assessment of B-cell and plasma cell frequencies in blood and bone marrow aspirates. Specimens were collected during pre-established study assessments or additional time points when collected as required for clinical management. Due to the challenges of assessing plasma cells, multiple methods were employed for their quantification in fresh specimens including flow cytometry and immunohistochemistry (IHC). Flow cytometric assessment of plasma cells was performed on freshly obtained marrow samples. Only patients with at least 3 months of B-cell aplasia in the absence of regular intravenous immunoglobulin (IVIg) infusions were included in the study. Results: All patients had no evidence of leukemia or peripheral B cells post-CART19 infusion at the time of this study. Compared to pre-CART19 serum titers, antibodies to S. pneumoniae remained stable or increased in 9 of 12 patients despite lack of circulating B-cells. Antibody titers to Tetanus toxoid were stable or increased in 13 of 14 patients. Anti-HIB levels were stable or increased in 9 of 11 patients and antibodies to Measles, Mumps and Rubella were stable or increased in 12 of 13, 11 of 13, and 12 of 13 patients, respectively. Flow cytometric analysis of bone marrow aspirates after CART19 infusion revealed three patients with persistence of CD38+ CD138+ plasma cells (at 1, 3 and 9 months post infusion, respectively) despite a complete absence of peripheral CD19+ B cells. In 9 patients, CD20 and CD138 IHC analysis of bone marrow core biopsies revealed a decrease in plasma cell (ranges: 1-5% pre-CART19, 0-<1% post-CART19), consistent with our previously published data. Finally, in another subset of patients, neither B cells nor plasma cells were detectable by flow cytometry of aspirate material or IHC of core biopsies collected either pre- or post-CART19 treatment. Conclusions: The stable or increased titers of antibodies to previous vaccines are surprising and may, in part, reflect improved marrow function as a result of leukemia eradication. The demonstration of plasma cells in a subset of patients in the absence of detectable tumor or normal B cells provides strong evidence for the existence of a population of plasma cells that are resistant to lysis by CART19 cells. This is consistent with antibody titers to previously generated vaccine antigens, which remain stable despite effective CART19 treatment. The additional finding of a decrease in CD138+ cells in several patients by IHC suggests that some populations of plasma cells are either targeted directly by CART19 or have a short half-life (e.g. plasmablasts); CD138 is not sufficient to distinguish these populations. Overall, these results indicate that long-lived plasma cells are resistant to CART19, likely due to a loss of CD19 during plasma cell differentiation. Continued analysis of remaining plasma cells in the absence of ongoing B-cell maturation as a result of CART19 persistence may provide important information on turnover rates of these long-lived cells in humans. Disclosures Bhoj: Novartis: Research Funding. Milone:Novartis: Patents & Royalties, Research Funding. June:Novartis: Research Funding, Royalty income Patents & Royalties. Porter:Novartis: Patents & Royalties, Research Funding. Grupp:Novartis: Research Funding. Melenhorst:Novartis: Research Funding. Lacey:Novartis: Research Funding. Mahnke:Novartis: Research Funding.

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 Rare case of non-producer variant of plasma cell dyscrasias with circulating plasma cells

2019 ◽  
Vol 12 (11) ◽  
pp. e231314
Author(s):  
Sukesh Manthri ◽  
Rabia Zafar ◽  
Robert Frank Cornell ◽  
Kanishka Chakraborty
Keyword(s):  
Bone Marrow ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Complete Response ◽  
Plasma Cell Neoplasm ◽  
Flow Cytometric ◽  
Plasma Cell Disorders ◽  
Plasma Cell Dyscrasias ◽  
Poor Outcomes ◽  
Marrow Cellularity

Non-producing variant of plasma cell disorders with circulating plasma cells is an aggressive variant of plasma cell dyscrasias with relatively poor outcomes. A 75-year-old man was admitted due to anaemia (90 g/L) and thrombocytopenia (9×109/L). Comprehensive metabolic panel showed creatinine of 1.34 mg/dL, total protein of 6 g/dL, and corrected calcium was normal. Peripheral smear review showed 8% circulating atypical plasmacytoid cells. Bone marrow biopsy (BMB) confirmed plasma cell myeloma involving 90%–95% of bone marrow cellularity. Serum protein electrophoresis showed no monoclonal protein. Due to aggressive biology of non-producer variant and outcomes based on circulating plasma cells, he was started on VD-PACE (bortezomib, dexamethasone, cisplatin, doxorubicin, cyclophosphamide and etoposide) chemotherapy. BMB after cycle 1 chemotherapy showed no morphologic, immunophenotypic, or flow cytometric features of a plasma cell neoplasm. Given excellent treatment response cycle 2 was changed to VRD (bortezomib, lenalidomide and dexamethasone). Following two cycles of VRD, he underwent autologous haematopoietic cell transplantation. Day 80 BMB suggested stringent complete response.

scholarly journals Multiple myeloma: circulating lymphocytes that express plasma cell antigens

Blood ◽  
1984 ◽  
Vol 64 (2) ◽  
pp. 352-356
Author(s):  
GJ Ruiz-Arguelles ◽  
JA Katzmann ◽  
PR Greipp ◽  
NJ Gonchoroff ◽  
JP Garton ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Plasma Cell ◽  
Peripheral Blood ◽  
Plasma Cells ◽  
Peripheral Blood Lymphocytes ◽  
Tumor Burden ◽  
B Cell Differentiation ◽  
Blood Lymphocytes ◽  
Bone Marrow Plasma

The bone marrow and peripheral blood of 14 patients with multiple myeloma were studied with murine monoclonal antibodies that identify antigens on plasma cells (R1–3 and OKT10). Peripheral blood lymphocytes expressing plasma cell antigens were found in six cases. Five of these cases expressed the same antigens that were present on the plasma cells in the bone marrow. Patients that showed such peripheral blood involvement were found to have a larger tumor burden and higher bone marrow plasma cell proliferative activity. In some patients, antigens normally found at earlier stages of B cell differentiation (B1, B2, and J5) were expressed by peripheral blood lymphocytes and/or bone marrow plasma cells.

scholarly journals Complement receptors regulate differentiation of bone marrow plasma cell precursors expressing transcription factors Blimp-1 and XBP-1

2005 ◽  
Vol 201 (6) ◽  
pp. 993-1005 ◽  
Author(s):  
Dominique Gatto ◽  
Thomas Pfister ◽  
Andrea Jegerlehner ◽  
Stephen W. Martin ◽  
Manfred Kopf ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Plasma Cell ◽  
Cell Activation ◽  
Plasma Cells ◽  
Antibody Responses ◽  
Complement Receptors ◽  
Essentially Normal ◽  
Bone Marrow Plasma

Humoral immune responses are thought to be enhanced by complement-mediated recruitment of the CD21–CD19–CD81 coreceptor complex into the B cell antigen receptor (BCR) complex, which lowers the threshold of B cell activation and increases the survival and proliferative capacity of responding B cells. To investigate the role of the CD21–CD35 complement receptors in the generation of B cell memory, we analyzed the response against viral particles derived from the bacteriophage Qβ in mice deficient in CD21–CD35 (Cr2−/−). Despite highly efficient induction of early antibody responses and germinal center (GC) reactions to immunization with Qβ, Cr2−/− mice exhibited impaired antibody persistence paralleled by a strongly reduced development of bone marrow plasma cells. Surprisingly, antigen-specific memory B cells were essentially normal in these mice. In the absence of CD21-mediated costimulation, Qβ-specific post-GC B cells failed to induce the transcriptional regulators Blimp-1 and XBP-1 driving plasma cell differentiation, and the antiapoptotic protein Bcl-2, which resulted in failure to generate the precursor population of long-lived plasma cells residing in the bone marrow. These results suggest that complement receptors maintain antibody responses by delivery of differentiation and survival signals to precursors of bone marrow plasma cells.

Expression of VEGF Receptors on Plasma Cells: Evidence of Heterogeneity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3361-3361
Author(s):  
Teresa K. Kimlinger ◽  
Thomas E. Witzig ◽  
S. Vincent Rajkumar
Keyword(s):  
Bone Marrow ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Receptor Expression ◽  
Vegf Receptors ◽  
Flow Cytometric Assay ◽  
Blocking Antibody ◽  
R And D ◽  
Flow Cytometric ◽  
Indirect Assay

Abstract Background: In previous studies quantitating VEGF receptors we have found no significant differences in expression between plasma cells from normal controls, multiple myeloma (MM), monoclonal gammopathy of undetermined significance (MGUS), or smoldering myeloma (SMM). (Kumar S, Blood, May 2004; 10.1182/blood-2003-11-3811 ePub). These studies were done using immunohistochemistry or Western Blotting (on CD138+ plasma cells) and may have been limited by low levels of receptor expression and by heterogeneity of expression. We measured expression of VEGF receptors (VEGFR1, VEGFR2 and VEGFR3) on the surface of plasma cells and in plasma cell subsets using direct and indirect flow cytometric assays to determine if significant differences in VEGF receptor expression existed between MGUS, SMM and MM. Methods: In the indirect flow cytometric assay, 32 bone marrow samples (3 amyloid, 9 MGUS, 12 MM, 8 SMM) were ACK lysed and tested using the FLUOROKINE TM rhVEGF biotin kit (R and D Systems, Minneapolis, MN) according to manufacturer’s instructions. In brief, in one tube (tube 1) cells were incubated with VEGF biotin. In tube 2, VEGF biotin preincubated with a blocking antibody was added to the cells (specificity control), while in a third tube a non-specific biotinylated protein (negative control) was added. After incubation, FITC-avidin, CD38 APC and CD45 Percp was added to each tube. Gates were drawn around the cells of interest and fitc staining was evaluated for the % positive cells. The % of signal blocked was calculated by comparing the fitc intensity (channel number) of the blocked VEGF peak (tube 2) to FITC intensity of tube 1. This system does not determine the identity of the receptor, but indicates the presence of VEGF receptors. In the direct flow cytometric assay, bone marrow from 25 individuals (2 amyloid, 5 MGUS, 7 MM, 7 SMM, and 4 normals) were lysed and blocked with mouse Ig and stained with CD38/CD45. In individual tubes, PE labeled VEGF R1, R2, R3 antibodies (R and D Systems, Minneapolis, MN) or isotype control were added. Plasma cells were identified, divided according to CD45 expression, and analyzed for % and intensity of receptor staining. Results: In the indirect assay, plasma cells in all groups bound VEGF ( 96% positive) at high intensity. There was also no difference in VEGF binding between CD45+ and CD45- plasma cell fractions (93 and 98% respectively).The specificity of VEGF binding (to one of the VEGF specific receptors) was confirmed by a significant drop in peak channel numbers of FITC intensity in the presence of blocking antibody. Specific VEGF binding at a similar intensity was seen in monocytes (95%) and at a lower intensity in lymphocytes (66%) and granulocytes (28%). Staining for VEGFR1, 2, and 3 in plasma cells using the direct assay revealed that except for 2 patients (1 amyloid and 1 SMM) none had >20% cells staining for any of the 3 receptors. The same results were seen in the CD45− fraction as well. In contrast, the CD45+ plasma cell fraction was highly positive for all 3 of the receptors in nearly all cases, with no significant differences between MGUS, SMM, amyloid, or MM. Conclusions: Plasma cells in MM and related disorders have specific VEGF receptors on the cell surface. The expression of VEGFR1, 2, and 3 seems to be primarily restricted to the CD 45+ subset of plasma cells. The finding of specific VEGF binding in CD45− plasma cells seen in the indirect assay may reflect the higher sensitivity of this assay due to the inbuilt amplification process or the presence of additional VEGF receptors such as neuropilin 1.

New Criteria To Identify Risk of Progression in Monoclonal Gammopathy of Uncertain Significance: Multiparametric Flow Cytometry Analysis of Bone Marrow Plasma Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3517-3517
Author(s):  
Ernesto Perez-Persona ◽  
María-Belén Vidriales ◽  
Gema Mateo ◽  
Ramón Garcia Sanz ◽  
Marivi Mateos ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Monoclonal Gammopathy ◽  
Plasma Cells ◽  
Flow Cytometry Analysis ◽  
Dna Index ◽  
Multiparametric Flow Cytometry ◽  
Uncertain Significance ◽  
Risk Of Progression ◽  
Bone Marrow Plasma

Abstract Monoclonal Gammopathy of Uncertain Significance (MGUS) is a monoclonal disorder defined by the presence of a serum monoclonal protein <3g/dL, bone marrow plasma cells < 10% and absence of end-organ damage. The risk of progression to multiple myeloma (MM) is about 1% per year, and therefore these patients require long follow-up. Accordingly, the definition of new parameters that could be used for the identification of patients at risk of progression could be of great value. The aim of the present study is to evaluate the utility of multiparameter flow cytometry analysis of bone marrow (BM) plasma cells (PC) for predicting the risk of progression of MGUS patients. From January 1996 to September 2004, bone marrow aspirate samples from 350 patients, who fulfil the criteria of MGUS according to the International Myeloma Working Group criteria, were analysed by multiparametric flow cytometry. A specific gate on PC was performed based on CD138/CD38 expression and FSC/SSC characteristics and PC were immunophenotypically classified as normal (polyclonal) or aberrant (clonal) according to the expression of CD138, CD38, CD45, CD19 and CD56 antigens. Twenty seven patients (8 %) progressed to MM, with a median time to progression (TTP) of 46 months (range 9 to 109 months). Interestingly, the percentage of aberrant PC within the total BM PC compartment (aPC/BMPCc) clearly identify patients at different risk of progression. Thus, TTP in patients with ≥ 95% aPC/BMPCc was 85 months vs not reached cases with <95% aPC/BMPCc (p=0.0000). Other parameters with a significant influence on progression in the univariate analysis were: paraprotein level (higher vs lower of 2 mg/dl; p= 0.0004), the presence of immunoparesis (no paresis vs. decreased levels in one or two Ig. p= 0.0005), Bence-Jones proteinuria (p= 0.0003), PC BM infiltration assessed both by morphology and flow cytometry (p=0.0074; and p= 0.001, respectively), and DNA index assessed by flow cytometry (diploid vs aneuploid; p=0.0064). Moreover, the cut off level of 95% aPC/BMPCc, also allows the discrimination of two risk categories upon considering only patients at low risk of progression, based on a low paraprotein level or absence of inmunoparesis (p= 0.0000 and p= 0.0000, respectively). On multivariate analysis only the percentage of aPC/BMPCc (≥95%) (p=0.000), the DNA index (p=0.007), and the Bence-Jones proteinuria (p=0.000) showed independent prognostic value. In summary, our results show that multiparameter FC evaluation of BMPC at diagnosis is a simple, cost-effective and valuable tool for predicting the risk of progression of MGUS patients.

Bone Marrow Immunophenotypic Analysis Allows the Identification of High Risk of Progression and Immune Condition-Related Monoclonal Gammopathy of Undetermined Significance.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2804-2804
Author(s):  
AndrÉs Jerez ◽  
Francisco Ortuño ◽  
María del Mar Osma ◽  
Ignacio Español ◽  
Ana Gonzalez ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
High Risk ◽  
Plasma Cells ◽  
Plasma Cell Dyscrasia ◽  
Newly Diagnosed ◽  
Immune Disorders ◽  
Chronic Infections ◽  
Risk Of Progression ◽  
Bone Marrow Plasma

Abstract Abstract 2804 Poster Board II-780 Background: Monoclonal gammopathy of undetermined significance (MGUS) progresses to plasma cell dyscrasia, mainly multiple myeloma (MM), at a rate of approximately 1% per year. Moreover, recent studies have shown that MM is nearly always preceded by MGUS, encouraging investigators to find better predictors for MM development in order to implement strategies to prevent or delay progression. In addition, a high prevalence of MGUS has been noted in a series of patients with immune disorders or chronic infections. Multiparameter flow cytometry allows the identification and quantification of both monoclonal and polyclonal plasma cells. This study analyses the relationship between monoclonal and polyclonal bone marrow plasma cells (BMPC), studied by means of flow cytometry, and its association with either immune or infectious disorders, or the development of MM in newly diagnosed MGUS patients. Methods: We conducted a retrospective cohort study to analyse the prognostic value of the aberrant (CD38++ CD138+ CD19– CD45weak) to normal (CD38++ CD138+ CD19+ CD45+) phenotype bone marrow plasma cells ratio (A/N ratio) and another 13 variables at baseline for the development of a plasma cell dyscrasia. We also performed a cross-sectional study to evaluate the association of those variables at baseline with the presence of a chronic immune response disorder. In each patient, the following variables were examined: age, sex, hemoglobin, serum creatinine, serum calcium, B2-Microglobulin, type and size of the serum monoclonal component (MC), isotype of the MC immunoglobulin, presence of urine MC, quantification of serum immunoglobulin levels, erythrocyte sedimentation rate, BMPC percentage and presence of atypical plasma cells on light microscopy, and aberrant and normal phenotype BMPC percentages. The effect of variables on progression was calculated using a Cox proportional hazards regression model. To identify variables at baseline associated with immune or chronic infectious disorders. a series of univariate and multivariate analyses was fitted using a binary logistic regression strategy. Results: Between March 1997 and April 2008, flow cytometry analysis on bone-marrow samples was performed on 322 patients with newly diagnosed MGUS. Median patient age was 71 years (interquartile range (IQR) 63-78 years) with a slightly male predominance (51%). Median follow-up was 46 months (IQR 23-58 months). During the period of observation, in 23 (7.1%) patients a transformation was registered into: MM (n=22), and primary amyloidosis (n=1). A total of 24 (7.4%) patients had a diagnosis of autoimmune disorder, and 18 (5.6%) patients of a chronic infection. Multivariate analysis for progression to MM revealed an increased A/N ratio as the main independent prognostic variable. In addition, our study found a significant association between a reduced A/N ratio and the diagnosis of a chronic immune response related condition. Using receiver-operating characteristic analysis we created an A/N ratio range from 4 to 0.20. Values of 4 or higher define a group of MGUS patients at high risk of progression (OR, 10.7; 95% confidence interval 4.2-39), whereas A/N ratio values of 0.20 or lower are associated with immune disorders or chronic infections (OR, 20.9; 95% confidence interval 8.5-51.1). A total of 282 patients had an A/N ratio below 4, and 42 had values equal to or above the cut-off. Patients with an A/N ratio ≥ 4 had a cumulative probability of transformation of 35% at 5 years, compared with 3% for those with an A/N ratio < 4. Conclusions: Extreme values of the A/N ratio at diagnosis seem to be related with two different conditions: high risk MGUS, likely to progress to MM, and immune condition related MGUS. Our findings further support the routine use of phenotypic characterization of bone marrow plasma cells in patients with MGUS at diagnosis. Disclosures: No relevant conflicts of interest to declare.

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