CD138-Independent Strategy for Detecting Residual and Circulating Myeloma Plasma Cells

Abstract INTRODUCTION: Flow cytometry has been used extensively to detect MM cells in the bone marrow (BM), micro-residual disease and circulating myeloma cells. Accumulating literature defines MM cells as CD138+/CD38+ for the primary gating of plasma cells; however, several studies demonstrated the presence of clonogenic CD138-negative MM cells, and that hypoxia can decrease the expression of CD138 in MM cells. We propose a novel set of biomarkers to detect MM cells regardless of their CD138 expression or hypoxic status. METHODS and RESULTS: We have tested the effect of hypoxia on the expression of the MM markers, CD138 and CD38, and found that hypoxia decreased the expression of CD138, therefore it cannot be used as a universal marker for MM cells. Hypoxia did not alter the expression of CD38 in MM cells; however, CD38 is a general leukocyte marker and cannot be used alone to identify MM cells, since it is expressed on multiple other leukocytes including T-cells, B-cells, monocytes, NK cells, and dendritic cells. Therefore, we negatively selected these cells by flow cytometry using the specific markers for each of these populations including CD3, CD19, CD14, CD16, and CD123; respectively. Therefore, we detected MM cells as CD38+/CD3-/CD19-/CD14-/CD16-/CD123-. We used CD38 antibody conjugated with APC and FITC-conjugated antibodies for all the other markers, thus MM cells were defined as APC+/FITC- population. To compare traditional method (CD138-based) with our strategy to detect hypoxic and normoxic MM cells, MM cell lines were stained with a cocktail of CD38-APC, FITC-antibodies, and CD138-V450, and analyzed by flow cytometry. The use of CD138+ as a universal marker for MM cells detected 85-100% of the normoxic cells, and only 60-75% of the hypoxic MM cells. While APC+/FITC- strategy detected close to a 100% of the MM cells independent of the cells’ normoxic/hypoxic status or expression of CD138. The ability of the new strategy to detect hypoxic and normoxic MM cells in the peripheral blood was tested by staining hypoxic and normoxic MM cells with cell-tracker Calcein-Red-Orange (as a positive control), spiked 104 MM cells into 106 mononuclear cells from a healthy donor (1% MM in total), and the percentile of Calcein-Red-Orange+ (as a positive control), APC+/FITC-, and CD138+ populations, were analyzed by flow cytometry. Calcein-Red-Orange staining showed exact 1% of MM cells detected in total mononuclear cells for both hypoxic and normoxic MM cells; detection with CD138+ showed 0.95% for normoxic and 0.45% for hypoxic cells; and detection of MM cells using the APC+/FITC- strategy showed 1.05% for normoxic and 1.1% for hypoxic MM cells. Hence, the new strategy detects MM cells selectively and independently of their CD138 expression or hypoxic status. We have used the APC+/FITC- strategy to detect MM cells in BM CD138-negative fractions of 20 MM patients. The APC+/FITC- strategy was able to detect a range of 1.6-44% myeloma cells in the CD138-negative population of BM mononuclear cells isolated from MM patients. Moreover, we have assessed the clonality of this population using APC+/FITC- in the CD138-negative fractions of MM patients. We found that the clonality of the APC+/FITC- population was similar to the clonality of the original disease (CD138+ cells) in 70% of the cases. The other 30% of the cases showed very low involvement of myeloma population in the BM and showed Kappa/Lambda ratio within normal range. Analyzing the prevalence of circulating MM cells in the peripheral blood from 12 MM patients showed that all patients had a higher number of circulating MM cells as detected by APC+/FITC- strategy compared to CD138+, and the fold change ranged from 1.5 to 86 times. CONCLUSION: We found that CD138 cannot be used as a universal marker to detect MM cells. Moreover, we developed a novel strategy to detect MM cells independent of their CD138 expression or hypoxic status; and we used CD38+/CD3-/CD19-/CD14-/CD16-/CD123- population as an alternative set of biomarkers to detect MM cells. This strategy was able to detect a clonal MM cell population in the CD138-negative fraction of BM mononuclear cells isolated from MM patients, as well as in the peripheral blood. Currently, we are exploring the ability of this strategy to predict relapse in MM patients whose BM was defined a CD138-negative. More investigation to characterize this population and the role in tumor recurrence and drug resistance in MM is warranted. Disclosures No relevant conflicts of interest to declare.

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Detection of Circulating Plasma Cells in Multiple Myeloma with Extramedullary Plasmacytoma

Blood ◽

10.1182/blood.v126.23.5328.5328 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5328-5328 ◽

Cited By ~ 1

Author(s):

Jing Wang ◽

Shuang Geng ◽

Yuping Zhong ◽

Mingyi Chen ◽

Wenming Wang ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Peripheral Blood ◽

Disease Surveillance ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Residual Disease ◽

Bone Marrow Aspiration ◽

Extramedullary Plasmacytoma

Abstract Objective: To detect the circulating plasma cells (cPCs) in patients of multiple myeloma (MM) with or withoutextramedullary plasmacytoma (EMP). Methods: The 21 patients of MM samples were collected from April 2014 to April 2015. There were 12 males, 9 females, with a median age of 60 (49 to 76 years old). Peripheral blood and bone marrow were examined before treatment or after EMP. Multi parameter flow cytometry (MFC) was used to analyze abnormal plasma cells (tumor cells) in samples of bone marrow and CD138 MACS positive sorting peripheral blood. The antibodies used in the flow cytometry were CD38-APC, CD138-PE, CD81-PE-Cy7, CD45-PacBlue, CD19-Percp-Cy5.5, CD56-mCherry-PE-ef610, CD117-AmCyan, CD16, Zombie-APC-Cy7. Results: In these 21 patients, he ratio of sex is 1.33:1, the median age is 60 (49-76). The immunoglobin type is as follows: IgG κ 7 cases, IgG λ 5 cases, IgG 2 cases, IgA κ 3 cases, IgA λ 2 cases, λ light chain 1 cases. The morphology of bone marrow aspiration showed more than 15% plasma cells and abnormal plasma cells can be seen in bone marrow in cytometry. 9 of 21 patients diagnosed MM with EMP, 2 of them find EMP when initial diagnosis and 7 of them find EMP in the course of disease (6 months to 8 years). The sites of the EMP included head, jaw, chest wall, side of the rib, the soft tissue of the sacral region and the vertebral body and all patients had bone involvement. In 17 patients with complete clinical data, bone marrow and peripheral blood specimens, the cPCs negative rate was 87.5%(7/8) in EMP negative patients, while the cPCs positive rate was 66.7% (6/9) in EMP positive patients, the difference among groups was statistically significant (chi square values: 5.13, p = 0.024). Conclusion: MFC has been widely used in diagnosis and minimal residual disease surveillance of MM, here we established the detection method of cPCs in MM patients, and it is valuable for clinical diagnosis and prognosis judgment. Disclosures No relevant conflicts of interest to declare.

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CS1: A Potential New Therapeutic Target for the Treatment of Multiple Myeloma.

Blood ◽

10.1182/blood.v108.11.3457.3457 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3457-3457 ◽

Cited By ~ 3

Author(s):

Eric D. Hsi ◽

Roxanne Steinle ◽

Balaji Balasa ◽

Aparna Draksharapu ◽

Benny Shum ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

B Cell ◽

Mononuclear Cells ◽

Plasma Cells ◽

Target Cells ◽

Dependent Manner ◽

Myeloma Cells ◽

Effector Cells

Abstract Background: To identify genes upregulated in human memory B and plasma cells, naïve B cell cDNA was subtracted from plasma cell and memory B cell cDNA. One gene that was highly expressed in plasma cells encodes CS1 (CD2 subset 1, CRACC, SLAMF7), a cell surface glycoprotein of the CD2 family. CS1 was originally identified as a natural killer (NK) cell marker. Monoclonal antibodies (mAbs) specific for CS1 were used to validate CS1 as a potential target for the treatment of multiple myeloma (MM). Methods: Anti-CS1 mAbs were generated by immunizing mice with a protein comprising of the extracellular domain of CS1. Two clones, MuLuc63 and MuLuc90, were selected to characterize CS1 protein expression in normal and diseased tissues and blood. Fresh frozen tissue analysis was performed by immunohistochemistry (IHC). Blood and bone marrow analysis was performed using flow cytometry with directly conjugated antibodies. HuLuc63, a novel humanized anti-CS1 mAb (derived from MuLuc63) was used for functional characterization in non-isotopic LDH-based antibody-dependent cellular cytotoxicity (ADCC) assays. Results: IHC analysis showed that anti-CS1 staining occurred only on mononuclear cells within tissues. The majority of the mononuclear cells were identified as tissue plasma cells by co-staining with anti-CD138 antibodies. No anti-CS1 staining was detected on the epithelia, smooth muscle cells or vessels of any normal tissues tested. Strong anti-CS1 staining was also observed on myeloma cells in 9 of 9 plasmacytomas tested. Flow cytometry analysis of whole blood from both normal healthy donors and MM patients showed specific anti-CS1 staining in a subset of leukocytes, consisting primarily of CD3−CD(16+56)+ NK cells, CD3+CD(16+56)+ NKT cells, and CD3+CD8+ T cells. Flow cytometry of MM bone marrow showed a similar leukocyte subset staining pattern, except that strong staining was also observed on the majority of CD138+CD45−/dim to + myeloma cells. No anti-CS1 binding was detected to hematopoietic CD34+CD45+ stem cells. To test if antibodies towards CS1 may have anti-tumor cell activity in vitro, ADCC studies using effector cells (peripheral blood mononuclear cells) from 23 MM patients and L363 MM target cells were performed. The results showed that HuLuc63, a humanized form of MuLuc63, induced significant ADCC in a dose dependent manner. Conclusions: Our study identifies CS1 as an antigen that is uniformly expressed on normal and neoplastic plasma cells at high levels. The novel humanized anti-CS1 mAb, HuLuc63, exhibits significant ADCC using MM patient effector cells. These results demonstrate that HuLuc63 could be a potential new treatment for multiple myeloma. HuLuc63 will be entering a phase I clinical study for multiple myeloma.

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Carfilzomib and Bortezomib Containing Regimens Yield High Rates Of MRD Negative Autologous Hematopoietic Progenitor Cell (HPC) Grafts In Multiple Myeloma (MM) and High Risk Smoldering Myeloma (SMM)

Blood ◽

10.1182/blood.v122.21.2030.2030 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2030-2030

Author(s):

Nishant Tageja ◽

Neha Korde ◽

Constance Yuan ◽

Kristen Cole ◽

Jennifer Hsu ◽

...

Keyword(s):

Flow Cytometry ◽

Stem Cell ◽

High Risk ◽

Tumor Cell ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Hematopoietic Progenitor Cell ◽

Myeloma Cells ◽

Cd34 Cells ◽

The Mean

Abstract Background Regimens incorporating modern anti-myeloma drugs, such as carfilzomib (CFZ) and bortezomib (BOR), produce rapid, deep and durable responses in newly diagnosed myeloma patients but their effect on collection of autologous HPC is not well known, including minimal residual disease (MRD) testing of stem cell grafts. Employing older induction regimens (such as VAD), less sensitive flow cytometry techniques detected circulating myeloma cells in 38-46% of autologous HPC grafts (Stewart, et al. JCO. 2001 and Bourhis, et al. Haematologica. 2007). We hypothesized that the use of modern CRd combination therapy including Carfilzomib (CFZ)-Lenalidomide (LEN)-Dexamethasone (DEX) would significantly lower the rates of HPC product contamination. Methods Thirty-six patients, including 29 with MM and 7 with high-risk SMM, underwent HPC mobilization and collection following induction with CRd (n=30), LEN-BOR-DEX (RVd, n=4), Cyclophosphamide-BOR-DEX (CyBorD, n=1) and Cyclophosphamide-BOR-Prednisone (CyBorP, n=1). For HPC mobilization, all patients received 5 days of filgrastim at 10-16 mcg/kg/dose. A combination of the patient’s weight and a peripheral blood CD34 count after 4 doses was used to determine the likelihood of collecting > 4 x106 CD34+ cells/ kg in a single apheresis procedure after a fifth filgrastim dose, according to a previously published algorithm from our institution. Only subjects predicted to require > 1 apheresis by the algorithm received Plerixafor (PLX) at 240 mcg/kg/dose on the fifth day along with the fifth filgrastim dose. HPC collection occurred on day 6, 8 hours after the last mobilizing agent(s) administration. Product contamination with myeloma cells (i.e. MRD status) was evaluated using multi-parameter flow cytometry with a minimum of 3 x 106 events obtained (sensitivity detection rate 1 x 10-5) to examine expression of 9 antigens by the plasma cells. Results The median age at mobilization was 56.2 years (range 40-73) and 19 (53%) were male. At the time of HPC collection, 20 (55%) patients were in sCR/CR/nCR, 11 (30%) had VGPR with 4 PR (11%) and 1 SD (3%). The mean CD34+ cells in the peripheral blood were 33/uL on day 5 and 55/uL on day 6 for the whole cohort. Thirteen (36%) patients did not need PLX. Interestingly, the mean CD34+ count dropped by a mean of 2% from D5 to D6 in patients not receiving PLX while, as expected, it increased by 304% in those who did. The median number of CD34+ cells collected was 6.86 million/kg (range 2.6-12.5) for the whole cohort, (6.6 million/kg without PLX and 7.52 million with PLX p=0.46). Thirty-three of 36 patients (92%) achieved a collection of > 4 million cells /kg in a single apheresis procedure. The 30 patients treated with CRd had a median of 5 (range = 3-7) prior cycles containing LEN with a median of 12 days (range 1-34) between mobilization and last LEN dose. Only 2 of 36 (5%) products were found to have evidence of tumor cell contamination (i.e. MRD positive) using sensitive multiparameter flow cytometry, one patient in PR after 6 cycles of CRd and a second patient in CR after 5 cycles of RVd. Conclusions Modern anti-myeloma therapies, such as CRd and RVd, allow adequate HPC collection in a single apheresis procedure in most cases and improve the quality of the HPC product with greatly reduced tumor cell contamination compared to historical controls. Indeed, 34/36 (94%) patients treated with modern anti-myeloma therapy collected an MRD negative HPC product. Future prospective studies are needed to assess whether autologous stem cell transplants (ASCT) using tumor-free HPC products collected in the era of modern induction therapies have better outcomes. Disclosures: No relevant conflicts of interest to declare.

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Myeloid-Derived Suppressor Cells in Patients with Hodgkin Lymphoma.

Blood ◽

10.1182/blood.v114.22.3662.3662 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3662-3662

Author(s):

Nunziatina Parrinello ◽

Piera La Cava ◽

Daniele Tibullo ◽

Cesarina Giallongo ◽

Orazio Di Bartolo ◽

...

Keyword(s):

Flow Cytometry ◽

Hodgkin Lymphoma ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Suppressor Cells ◽

Cell Number ◽

Myeloid Derived Suppressor Cells ◽

Peripheral Blood Mononuclear ◽

Hla Dr

Abstract Abstract 3662 Poster Board III-598 Background Immune suppression and angiogenesis are mechanisms key to tumour growth and progression. Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells of myeloid origin and include immature macrophages, dendritic cells (DC) and other myeloid cells. In mice are phenotypically characterized as CD11b+Gr-1+ cells, while in human they have an immature phenotype, including lineage negative (Lin-), CD14-, HLA-DR-, CD15+, CD34+, CD11b+, CD33+, and CD13+ cells. MDSC reduce activated T-cell number and inhibit their function through different mechanisms including: L-arginine metabolism, nitric oxide (NO), up-regulation of reactive oxygen species (ROS), and secretion of immunosuppressive cytokines. MDSC also promote tumor-dependent angiogenesis as well as tumor metastasis. Their accumulation has been described in patients affected by some solid tumors but information on haematological neoplasms are lacking. Our study investigated by flow cytometry the presence of MSDC in the peripheral blood of patients affected by Hodgkin Lymphoma (HL). Methods We studied 14 patients with HL at diagnosis and 10 age-matched healthy controls (HC). Peripheral blood mononuclear cells were stained with the following monoclonal antibodies:CD11b, CD13, CD14, CD34, CD45, for 20 minutes at room temperature. After lysing red cells, cells were analyzed by flow cytometry. Results we observed a increased number of MDSC (CD11b+,CD13+,CD34+,CD14-, CD45+) in the peripheral blood of patients with HL compared to HC (13,37 ± 17,77 ×109/l vs 1,45± 0,98 ×109/l, p=0,0007). We also found that patients with advanced-stage Hodgkin disease (III and IV) have higher number of MDSC, compared to patients stage I and II (p= 0,04). Conclusion These data suggest a role for myeloid-derived suppressor cells in promoting tumor cell proliferation in hodgkin lymphoma. Disclosures: No relevant conflicts of interest to declare.

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Role of NKG2D in Cytokine-Induced Killer (CIK) Cells Against Multiple Myeloma Cells

Blood ◽

10.1182/blood.v118.21.5119.5119 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5119-5119

Author(s):

Xu-zhang Lu ◽

Bao-An Chen ◽

Lin-di Ma ◽

Xiao-hui Cai ◽

Min Zhou

Keyword(s):

Flow Cytometry ◽

Mononuclear Cells ◽

Plasma Cells ◽

Nk Cell ◽

Conflicts Of Interest ◽

Human Peripheral Blood ◽

Nkg2d Ligands ◽

Cik Cells ◽

Ifn Γ ◽

Nkg2d Receptor

Abstract Abstract 5119 Cytokine-induced killer (CIK) cells are T lymphocytes enriched in CD3+CD56+ cells, which can be easily and rapidly expanded in vitro from human peripheral blood, bone marrow or cord blood mononuclear cells with the sequential addition of interferon (IFN)-{gamma}, OKT-3 and high doses of interleukin (IL)-2. The cytokine-induced killer (CIK) cells have been reported to have potent cytotoxicity against a variety of tumor cells including multiple myleoma (MM) cells. The mechanism of CIK cell recognizing MM cells remains unknow. Recent studies indicated that ligation of NKG2D on immunological cells directiy induce cytotoxicity. We suspect whether NKG2D receptor induction on CIK cells by cytokines is responsible for the killing of MM cells by CIK. We expended CIK cells from healthy controlswith interferon (IFN)-γ, CD3 monoclonal antibodies (mAb) and IL-2, and checked expression of NK cell receptors on CIK cells by flow cytometry. We found higher expression of NKG2D receptor and lower other NK receptors, such as CD158a,CD158b and NCRs on expanded CIK. These CIK cells showed higer cytotoxicity to multiple myleoma cell line U266 expressing NKG2D ligands. Interestingly, when cocultured with U266 cells, only NKG2D expressing CIK cells released IFN-γ detected by flow cytometry. We next analyzed NKG2D ligands expression on primary plasma cells in 22 MM patients by flow cytometry, the primary plasma cells in 16/22 (72.7%) MM patients expressed different levels of ULBPs or MICA/B on the cell surface. CIK cells showed higher cytotoxicity (12.5%) to NKG2D ligands expressing primary plasma cells compared to those did not express NKG2D ligands. The killing of CIK against MM cells were partially blocked by treatment of CIK with anti-NKG2D antibody. We conclude that NKG2D-NKG2D ligangd interaction may be one of the mechanisms by which CIK cells kill MM cells. Disclosures: No relevant conflicts of interest to declare.

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CD19 Negative Relapse in B-ALL Treated with Blinatumomab Therapy: Avoiding the Trap

Blood ◽

10.1182/blood.v126.23.4983.4983 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4983-4983 ◽

Cited By ~ 7

Author(s):

Costas K Yannakou ◽

Neil Came ◽

Ashish R Bajel ◽

Surender Juneja

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Residual Disease ◽

Single Agent ◽

Normal Bone Marrow Cell ◽

Leukaemic Cells ◽

Antibody Panel ◽

B Lineage

Abstract Acute lymphoblastic leukaemia of the B lineage (B-ALL) is an aggressive neoplasm of B lymphocyte precursors that expresses the pan B cell marker CD19 in the majority of cases. For this reason CD19 has played a pivotal role as a 'gating' antigen in the analysis of leukaemic cells by flow cytometry, both at diagnosis and during minimal residual disease (MRD) monitoring. In recent years, novel therapeutic strategies have been developed for the treatment of B-ALL. Blinatumomab, a bispecific T cell engager (BiTE®) antibody and chimeric antigen receptor-modified T cells (CARTs) are treatments with targeted anti-CD19 activity that have shown promising efficacy in clinical trials. Despite the burgeoning promise of targeted anti-CD19 strategies a concerning number of CD19 negative relapses have been reported in this setting - this argues strongly for down regulation of CD19 by the leukaemic clone or selection of pre-existing CD19 negative clones as mechanisms of therapy resistance. The trials published to date have used PCR-based methods for MRD monitoring which has allowed for early detection of molecular relapse, with CD19 status being assessed by flow cytometry after morphological relapse. Outside of the trial setting flow cytometry based MRD monitoring is regularly used. This poses a major practical issue for institutions that treat B-ALL patients with targeted anti-CD19 therapies as conventional CD19 gating based B-ALL MRD panels are insensitive to the presence of CD19 negative leukaemic cells. We present the case of a 69 year old male with relapsed CD19 positive B-ALL who attained complete immunophenotypic remission after one cycle of single agent blinatumomab. He then relapsed with CD19 negative disease as detected by an alternative gating strategy within the Clinical Oncology Group-based antibody panel used routinely for B-ALL assessment at our institution. B-ALL MRD analysis by flow cytometry at our institution involves the collection of whole bone marrow in sodium heparin, erythrocyte lysis and analysis on a Beckman Coulter NaviosTM flow cytometer within 24 hours using the following single tube antibody cocktail: CD45-KO, CD20-PacBlue, CD38-APC750, CD19-APC700, CD58-APC, CD13+33-PC7, CD56-PC5.5, CD34-ECD, CD10-PE and CD9-FITC. Kaluza® analysis software is used to define viable cells by excluding doublets, erythrocytes and cellular debris followed by the gating of CD19+ cells, excluding plasma cells and comparing the expression of other antigens to normal B-lineage maturation patterns. B-ALL is defined as a cluster of viable CD19+ cells that do not conform to normal B-lineage maturation patterns in multiple dimensions, typically: CD20vsCD10, CD38vsCD34, CD45vsCD34, CD45vsCD10, CD45vsCD38+/- aberrant CD9, CD13+33 or CD58 expression. Following therapy in this case and in the absence of detectable CD19+ cells, gating was based on first identifying known major normal bone marrow cell populations using known antigen expression and location on the CD45/side scatter(SSC) plot: mature lymphocytes (CD45-high/SSC-low), granulocytes (CD10+/SSC-high), plasma cells (CD38++) and myeloblasts (CD34+/CD45+/13+33+/SSC-medium). In this case B-ALL cells were identified as CD34+/SSC-low, residing outside of expected regions for normal CD34+ B-lymphoid precursors in dimensions not dependant on CD19. This finding was followed by morphological relapse within 3 months. This case highlights the potential limitations of CD19 gating for B-ALL MRD assessment by flow cytometry in patients treated with targeted anti-CD19 therapies. It demonstrates an alternative algorithm that may assist diagnostic laboratories with limited access to PCR-based methodologies by using an already widely available antibody panel. It is acknowledged that in this case the retained expression of CD34 by the leukaemic B-lymphoblasts was pivotal in tracking the leukaemia after therapy. In order to address the possibility of CD34/CD19 dual negative disease, the use of alternative B-lineage gating and/or maturation antigens such as CD22 and CD81 is being evaluated. Awareness of this important issue and the need for an adapted approach to gating in this clinical setting will become increasingly important as targeted therapies become more widely adopted for the treatment of B-ALL. Disclosures No relevant conflicts of interest to declare.

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Interphase Chromosome Flow-FISH

Blood ◽

10.1182/blood.v120.21.4925.4925 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4925-4925

Author(s):

Jason Weed ◽

Keyvan Keyvanfar ◽

Prashanth Swamy ◽

Sachiko Kajigaya ◽

Rodrigo T. Calado ◽

...

Keyword(s):

Flow Cytometry ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Chromosome 7 ◽

Interphase Chromosome ◽

Monosomy 7 ◽

Disease Characteristics ◽

Conventional Cytogenetics ◽

Cellular Dna

Abstract Abstract 4925 We have developed a method combining fluorescence in situ hybridization (FISH) and flow cytometry to detect monosomy 7 in interphase cells of patients with myelodysplastic syndrome. This method, Interphase Chromosome Flow-FISH (IC Flow-FISH), involves fixation of leukocytes cells, permeabilization of cellular membranes, DNA hybridization with peptide nucleic acid (PNA) probes, and analysis by flow cytometry. Samples of 10–20 mL of peripheral blood in patients with monosomy 7 yielded hundreds to thousands of monosomy 7 cells detected by flow cytometry. Peripheral blood samples were obtained from patients with MDS who tested positive for monosomy 7 by conventional cytogenetics, and mononuclear cells were isolated by Ficoll density centrifugation and fixed with Carnoy's solution. Cell membranes were then permeabilized with a saponin-based detergent, and cellular DNA was denatured with heat and hybridized with denatured PNA probes while the cell membrane remained intact. Next cells were washed and analyzed by flow cytometry. Healthy donor samples showed similar distributions of chromosome 7 probe fluorescence between monocytes and (unaffected) lymphocytes such that there was no clear distinction between these populations in a histogram of probe fluorescence (fig 1A). In samples from patients with monosomy 7, a population of monocytes exhibited distinctly lower probe fluorescence than did normal lymphocytes and monocytes (fig 1B). As MDS affects cells of the myeloid rather than the lymphoid lineage, a decrease in probe binding among monocytes rather than lymphocytes was consistent with disease characteristics. In 5 patient samples, there were no statistically significant differences between proportions of monocytes with monosomy 7 detected in IC Flow-FISH and proportions of cells with monosomy 7 detected in conventional cytogenetics. Patient cells were sorted with fluorescence-activated cell sorting (FACS) to verify the population of cells with lower probe fluorescence corresponded to cells having monosomy 7 and that cells with higher fluorescence showed normal chromosome 7 copy number. Of patient cells gated for lower fluorescence, 45 of 50 (90%) cells analyzed by microscopy showed 1 distinct nuclear signal and 5 of 50 (10%) showed 2 signals. Of cells gated as having higher fluorescence, 39 of 42 (93%) cells showed 2 distinct signals and 3 of 42 (7%) showed only 1 distinct signal. IC Flow-FISH provides the ability to detect monosomy 7 in MDS patients without bone marrow procurement or metaphase spreads. Because the number of monosomy 7 cells isolated by FACS may range in the thousands, the method offers a large amount of data while also allowing automated detection and isolation of cells with aneuploidy for further analysis and characterization. Application to other chromosome number abnormalities has the potential to expand the range of the technique to a variety of hematologic disease settings. Figure 1 Figure 1. Disclosures: No relevant conflicts of interest to declare.

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Study On Immune Mechanism of Human Marrow Mesenchymal Stem Cells Adjusting Dendritic Cells for Treatment of Aplastic Anemia

Blood ◽

10.1182/blood.v116.21.5137.5137 ◽

2010 ◽

Vol 116 (21) ◽

pp. 5137-5137

Author(s):

Yang Xiao ◽

Leqin Zhang

Keyword(s):

Flow Cytometry ◽

T Lymphocytes ◽

Surface Antigen ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Human Peripheral Blood ◽

Interleukin 4 ◽

Peripheral Blood Flow ◽

Inhibiting Effect

Abstract Abstract 5137 Objective (1)To explore whether MSC has inhibiting effect on the proliferation ofAA patients' Tcell;(2)To discuss whether MSC affects T cell's proliferation via adjusting the growth of DCs. Materials and Methods (1) MSC were separated and cultured in vitro. Cell morphology was observed and the cell surface antigen was determined by flow cytometry.(2) Peripheral blood mononuclear cells were extracted from 20 patients suffered AA and then T lymphocytes were separated by nylon fiber column. Flow cytometry was applied to determine the surface antigen and subpopulation of T cell.(3)mononuclear cells were separated from normal human peripheral blood. DCs were prepared under the culture condition of recombination human granulocyte-macrophage colony stimulating factor (GM-CSF) and recombinant human interleukin-4 (IL-4). After acquiring the mature DCs induced by LPS, the phenotype analysis of DCs before and after culture was examined by flow cytometry, respectively. Results (1) After co-culture of MSC and T lymphocytes from AA peripheral blood, flow cytometry showed that the ratio of D8+ in T cells reduced significantly from 38.7% to 29.7 % (p < 0.05), whereas the CD4+ ratio increased from 24.9% to 34.9% significantly (p < 0.05). Meanwhile, ELISA analysis indicated that the concentration of IL-2 and IFN-γ were significantly decreased from 38.9 and 38.5 ng/L to 6.8 and 6.6 ng/L, respectively (p < 0.05). However, IL-4 and IL-10 increased from 2.8 and 2.9 to 5.3 and 8.3 ng/L, respectively (p < 0.05).(2) After the induction of immature DCs by LPS, flow cytometry showed that the expression of CD1a increased from 2.4% to 68.4% in the treatment without MSC, while that of CD14+ decreased from 83.6% to 3.5% (p < 0.05).(3) After the co-culture of mature DCs and MSC, the expression of CD14+ increased from 5.8% to 62.8% when the expression of CD1a, CD83 and CD80 decreased from 48.6%, 60.8% and 50.2% to 30.7%,40.9% and 20.3%, respectively. Conclusions Our study shows that (1)MSC inhibits the proliferation of T lymphocytes from AA p-atients by regulating CD8+ and CD4+; (2)futher study indicates that MSC can inhibit the growth of DCs and reverse the status of DCs from matureness to immatureness; (3)thus suggests the possible mechanism of MSC's inhibiting effect as follows: MSC decreases the liveness by controlling the growth of DCs and further inhibits its proliferation. Disclosures: No relevant conflicts of interest to declare.

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The effect of a leukodepletion model on the activation stage of platelets

Open Medicine ◽

10.2478/s11536-010-0062-1 ◽

2011 ◽

Vol 6 (2) ◽

pp. 181-184

Author(s):

Miodrag Vucic ◽

Ivan Tijanic ◽

Nenad Govedarevic ◽

Lana Macukanovic ◽

Zoran Pavlovic

Keyword(s):

Flow Cytometry ◽

Proinflammatory Cytokines ◽

Mononuclear Cells ◽

Buffy Coat ◽

The Other ◽

Therapeutic Doses ◽

Cell Counter ◽

Automatic Cell Counter

AbstractThe preparation of thrombocyte concentrates with filtration before storage (in-line) makes it possible to avoid the presence of mononuclear cells in the concentrate and proinflammatory cytokines. Therefore, this filtration may result with decreased activation of trombocyte receptors in vitro, which may improve therapeutic efficiancy. Methods. We compared two groups, each with 30 therapeutic doses of concentrated thrombocytes. We prepared the first group using the classic model from the buffy coat and the other with concentrated thrombocyte samples filtrated during sampling, so-called in-line, with the WBC filter Imuflex (Terumo). Mononuclear cells (MNC), thrombocyte, and erythrocyte counts in the units of concentrated thrombocytes were obtained on an automatic cell counter, and we used flow cytometry to measure the expression of surface thrombocyte receptors. The results demonstrated that the trombocytes prepared with pre-storage filtration contained a very low level of mononuclear cells and markedly reduced trombocyte receptors. Conclusion. The number of MNC and expression of surface thrombocyte receptors were markedly lower in the concentrated thrombocyte units prepared with in-line filtration. The thrombocytes prepared in this way contain fewer mononuclear cells, are of higher quality, are more functional, and may produce a better therapeutic effect in vivo.

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Platelet and Immune Characteristics of Patients with Immune Thrombocytopaenia Non Responders to Therapeutic Treatments

Blood ◽

10.1182/blood-2019-125772 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1089-1089

Author(s):

Elena Monzón Manzano ◽

Raul Justo Sanz ◽

Diana Hernández ◽

Teresa Álvarez Roman ◽

Ihosvany Fernandez-Bello ◽

...

Keyword(s):

Flow Cytometry ◽

Immune Response ◽

Sialic Acid ◽

Research Funding ◽

Caspase 3 ◽

Mononuclear Cells ◽

Ricinus Communis ◽

The Other ◽

Control Group ◽

Platelet Surface

Introduction: Mechanisms leading to diminished platelet counts in immune thrombocytopaenia (ITP) appear to be multifactorial: autoantibodies, autoreactive CD8+ cytotoxic T cells, enhanced apoptosis and loss of sialic acid which mediates platelet clearance through the Ashwell-Morell receptors present in hepatocytes. Differential involvement of each of them might condition the ability of patients with ITP to respond to treatments. We aimed to examine platelet features and the immunological state of patients with ITP who do not respond to any treatment to detect the unique characteristics of this group. Methods: This was an observational, prospective and transversal study. Patients with chronic primary ITP were included: 28 ITP patients without treatment for at least 6 months (UT-ITP); 36 responders to agonists of thrombopoietin receptors (TPO-RA); and 14 ITP patients who did not respond to first- and second-line treatments (NR-ITP). A healthy control group (n=104) was also included in the study. Active caspase-3, -7, -8 or -9 were determined by flow cytometry using CaspaTag kits (Millipore, Madrid, Spain) in PRP diluted with HEPES-buffer containing 2 mM Ca2+ and 2 mM Gly-Pro-Arg-Pro (Sigma-Aldrich, Madrid, Spain) to prevent fibrin formation . Platelet surface glycan exposure was analysed by determining the binding of lectins by flow cytometry. To do so, washed platelets were incubated with 1 μg/ml Alexa fluor 488-conjugated wheat germ agglutinin lectin (WGA, Invitrogen, Spain) or with 1 μg/ml FITC-conjugated Ricinus communis agglutinin (RCA, Vector Labs, UK). WGA binds to sialic acid and N-acetylglucosaminyl residues, and RCA is a galactose-specific legume lectin which binding serves as an indirect measurement of the loss of sialic acid. Peripheral blood mononuclear cells (PBMCs) subsets were analysed by flow cytometry using specific antibodies. Experimental data was analysed using SPSS 9.0 software (SPSS Inc., Chicago, IL). Results: Platelets from TPO-RA treated and from NR-ITP patients had increased caspase-3, -7, -8 and -9 activities (Figure 1A). Platelets from NR-ITP patients exposed less sialic acid and more N-acetylglucosaminyl residues than the other groups (Figure 1B). Binding of WGA and RCA correlated with caspase activities (Table 1). Distribution of lymphocytes, monocytes and natural killer cells is shown in Table 1. NR-ITP patients had an increased proportion of B lymphocyte (LB), maybe due to a significant rise in the fraction of naive LB cells, and a diminution in LTreg subset. Whereas classical monocytes was increased, nonclassical monocyte fraction was decreased in the UT-ITP and NR-ITP groups. NR-ITP patients also presented an increased CD16+CD56bright cells fraction and a diminished NK CD16+CD56dim subset. TPO-RA-treated patients seemed to recover an immune homeostasis similar to healthy controls (monocyte and NK cells subset distribution and LTreg count similar to control group). It is of interest to note the relationship between loss of sialic acid from platelet surface glycans and Tregs count: the most reduced surface exposure of sialic acid, the less Treg count (Figure 2). Conclusions: Platelets from NR-ITP patients had more signs of apoptosis and a different composition of surface glycans, accompanied by a diminished LTreg population, a higher LB naïve percentage, and an increased CD16+CD56bright cells fraction in circulation, indicating a severe deregulation of the immune system. Since an inverse correlation was observed between loss of sialic acid and LTreg count, a potential relationship between glycan composition on the platelet surface and immune response is suggested, positing terminal sugar moieties of the glycan chains as aetiopathogenic agents in ITP. On the other hand, TPO-RA appears to have a beneficial effect on immune response. Nevertheless, one of the limitations of our study was that patients were recruited once the response to TPO-RA was achieved; therefore, a longitudinal study would provide more information regarding TPO-RA effects. This work was supported by grants from the FIS-FONDOS FEDER (PI15/01457, NB). NVB holds a Miguel Servet tenure track grant from FIS-FONDOS FEDER (CP14/00024). Disclosures Álvarez Roman: Roche: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Bayer: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Takeda: Research Funding; NovoNordisk: Consultancy, Speakers Bureau; CSL Behring: Consultancy, Speakers Bureau; Sobi: Consultancy, Speakers Bureau. Fernandez-Bello:Novartis, Pfizer, ROCHE, Stago: Speakers Bureau. Martín:SOBI: Research Funding; Novartis, Pfizer, ROCHE, Novo Nordisk: Speakers Bureau. Rivas Pollmar:Novartis, Pfizer, ROCHE, Novo Nordisk: Speakers Bureau; SOBI: Research Funding. Canales:Novartis: Honoraria; Takeda: Speakers Bureau; iQone: Honoraria; Sandoz: Honoraria; Celgene: Honoraria; SOBI: Research Funding; Karyopharm: Honoraria; F. Hoffmann-La Roche Ltd: Honoraria, Speakers Bureau; Gilead: Honoraria; Janssen: Honoraria, Speakers Bureau. Jimenez-Yuste:Bayer, CSL Behring, Grifols, Novo Nordisk, Octapharma, Pfizer, Roche, Sobi, Shire: Consultancy, Honoraria, Other: reimbursement for attending symposia/congresses , Research Funding, Speakers Bureau. Butta:Novartis: Consultancy; Roche, Pfizer: Speakers Bureau.

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