Family-Associated Monoclonal B Lymphocytosis Shows Differences From CLL That Suggest An Indolent Biology.

Abstract Abstract 1241 Poster Board I-263 Background and Significance Monoclonal B lymphocytosis (MBL) is a hematologic syndrome characterized by accumulations of clonal B lymphocytes in the peripheral blood. Most MBL have a CLL-like immunophenotype, though other, less common phenotypes are observed. Although some MBL, particularly those with MBL cell counts >1.9 × 109 / L, progress to CLL, “low count” MBL (<0.2 × 109 MBL / μL) have little potential to progress to MBL and may have different biologic characteristics from MBL with lymphocytosis or Rai Stage 0 CLL. Our hypothesis was that detailed characterization of family-associated MBL may also provide biologic insights into the pathogenesis of CLL. Methods Individuals with MBL were identified by flow cytometry screening of fresh and cyropreserved peripheral blood from unaffected members of CLL kindreds ascertained by Genetic Epidemiology of CLL Consortium (GEC) sites. We defined MBL as populations of CD5+, CD19+, CD20lo, CD23+ B cells that comprised at least 0.02% of the PBMC and did not exceed 5.0 × 109 MBL cells / L. Flow cytometry was used to determine the surface immunophenotype including prognostic parameters of CD38, intracellular ZAP-70, immunoglobulin isotype, CD49d, and the ratio of CD69:71. mRNA and genomic DNA from single MBL cells isolated by flow cytometric sorting were analyzed using PCR to determine immunoglobulin heavy and light chain sequences (IGVH status). MBL cells were sorted in bulk for FISH determination of genetic loci associated with clinical CLL. Results Fifty-four unaffected family members were found to have MBL, of these 48 (89%) showed a typical CLL immunophenotype (CLL-like MBL). We observed significant variability in the size of the MBL clone as a percentage of the CD19+ B cell compartment (mean 30%, median 16%; range 2% - 97%). CD38 positive (defined as CD38 surface expression in ≥ 30% of MBL cells) was observed in 6 of 38 (16%) subjects tested. ZAP-70 positive (defined as intracellular expression in ≥ 20% of MBL cells) was expressed in 5 of 28 (18%) participants. CD49d positive (defined as surface expression in ≥ 45% of MBL cells) was observed in 4 of 17 (24%) subjects. The ratio of surface expression of CD69:CD71, a measure of cellular activation that also correlates with an unmutated IGVH, was ≥2.0 in 3 of 34 (9%) subjects tested. Among 36 subjects tested, 9 (25%) MBL expressed both surface IgD and IgM (defined as surface expression ≥ 40% for both IgM and IgD), 12 (33%) expressed IgD only, 2 (6%) expressed IgM only, and 13 did not express IgD or IgM (36%). Analysis of IGVH status has been completed in 10 individuals. Both immunoglobulin heavy chain variable (IGVH) region mutated (n = 14) and unmutated (n = 5) sequences were observed. Six of 10 individuals had 2 or more unrelated MBL clones (range 2 - 4), including two individuals with both unmutated and mutated clones. Among the 19 MBL clones identified in these 8 subjects, VH3 or VH4 rearrangements were observed in all MBL clones. The most commonly rearranged IGVH genes were 3-07 (3 MBL clones), 3-15 (3), and 4-34 (3). No VH1 family gene rearrangements were observed. MBL cells were bulk sorted for FISH from 14 subjects. Mono or biallelic deletion of 13q14.3 was observed in 9 subjects, 4 were normal, and one showed trisomy 12. Conclusions Our data affirms that CLL-like MBL are commonly observed among the unaffected family members from CLL kindreds. We found that family associated MBL clones (most of which were small clones) express ZAP-70, CD38, and CD49d at an apparently lower frequency than observed in CLL. Unlike in CLL, the surface immunoglobulin isotype showed co-expression of IgM and IgD in only one third of cases. Moreover, small MBL clones are commonly oligoclonal and predominantly express mutated IGVH genes with an IGVH usage that also appears different from CLL. Taken together, these findings suggest that small MBL clones, though phenotypically similar to CLL, have important biologic differences from CLL that may explain the limited potential of these clones to progress to CLL. The clinical outcome of these MBL clones in relation to our baseline prognostic characterization will be of interest. In addition the further investigation of family associated MBL is being conducted and may clarify the genetics and immunobiology of familial CLL. Disclosures No relevant conflicts of interest to declare.

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Leukocyte Integrin CD18 Expression Mediates Transient Neutropenia Following G-CSF Administration.

Blood ◽

10.1182/blood.v114.22.3587.3587 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3587-3587

Author(s):

Laura Tuschong ◽

Catherine E. Dejesus ◽

Meredith Adams ◽

Aylin C. Bonifacino ◽

Dennis D. Hickstein ◽

...

Keyword(s):

Peripheral Blood ◽

Conflicts Of Interest ◽

Leukocyte Adhesion ◽

Surface Expression ◽

Peripheral Blood Leukocytes ◽

Blood Leukocytes ◽

Cell Counts ◽

Transient Loss ◽

The Mean ◽

White Blood Cell Counts

Abstract Abstract 3587 Poster Board III-524 The mechanism whereby neutrophils traffic from the circulation in response to G-CSF has remained unclear despite the observation of ourselves and others that there is a dramatic, yet transient, loss of circulating neutrophils shortly following the administration of G-CSF in humans, non-human primates, and mice (Gordon BC, et al. Exp Hematol. 35:872-8, 2007). To determine the role of the CD18 leukocyte integrin on neutrophils in the egress of neutrophils from the circulation, we used dogs with canine leukocyte adhesion deficiency (CLAD), a genetic disease in which a mutation in CD18 prevents CD18 surface expression. We selected CLAD dogs who had 5-10% CD18+ neutrophils following either matched littermate allogeneic transplant or autologous gene therapy for CLAD. Three CLAD dogs meeting these criteria were evaluated. Three carrier dogs served as controls. G-CSF was administered at 10μg/kg SQ to all six animals. Peripheral blood samples (EDTA) were taken immediately prior to G-CSF administration, and at 15, 30, 60, 120, 240 minutes, and 24 hours following G-CSF administration. Total white blood cell counts, neutrophil counts, and the number and percentage of CD18+ peripheral blood leukocytes were assessed. As anticipated, the control dogs had a 60% decrease in circulating neutrophils 30 minutes following G-CSF administration: the mean +/− standard of deviation (SD) absolute neutrophil baseline count decreased from 6806+/−1072/μL to 2727+/−767/μL. In five control animals the neutrophil nadir occurred at 30 minutes post-G-CSF, and in one control dogs it occurred 15 minutes following G-CSF administration. Experimental CLAD dogs had only a 35% decline in neutrophil numbers at 30 minutes, from a mean baseline of 6777+/− 672/μL to 4433+/−265/μL. In these dogs the neutrophil count returned to pre-G-CSF levels by 60 minutes post-G-CSF. By 24 hours after G-CSF, the neutrophil level was increased 3-fold from baseline. Immunophenotyping using an anti-CD18 and a canine specific anti-neutrophil PE conjugated antibody indicated that only the CD18+ neutrophils disappeared from the circulation following G-CSF administration. At baseline the transplanted CLAD dogs had a mean of 15.2+/−3.9% CD18+ peripheral blood leukocytes, of these 50.7+/−7.1% were CD18+ neutrophils. Thirty minutes following G-CSF administration the mean+/−SD percentage of CD18+ leukocytes declined to 13.7+/−3.7% with 33.4+/−5.8% being neutrophils. There was also a slight decline in CD14+CD18+ monocytes from 6.2 +/− 1.5% to 4.0 +/− 1.2%, which was not observed in the controls. There was no change in CD18- leukocyte numbers. The percentage of CD18+ neutrophils returned to baseline by 60 minutes and remained there at subsequent time points. These results demonstrate that the CD18 leukocyte integrin on circulating neutrophils mediates the transient neutropenia associated with G-CSF administration. Disclosures: No relevant conflicts of interest to declare.

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The Role of Surface Immunoglobulin Isotype in Chronic Lymphocytic Leukemia Disease Biology and Clinical Outcome

Blood ◽

10.1182/blood.v118.21.2850.2850 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2850-2850

Author(s):

Danielle M. Brander ◽

Sallie D. Allgood ◽

Karen M. Bond ◽

J. Brice Weinberg ◽

Mark C Lanasa

Keyword(s):

Flow Cytometry ◽

B Cells ◽

Conflicts Of Interest ◽

Fold Increase ◽

Surface Expression ◽

Lymphocytic Leukemia ◽

Mutation Status ◽

Immunoglobulin Isotype ◽

Surface Immunoglobulin

Abstract Abstract 2850 Background: Upon encountering cognate antigen, naïve B cells may undergo germinal center reaction. This results in DNA modification including somatic hypermutations (HSM) of the immunoglobulin heavy chain variable region gene (IGHV) and immunoglobulin class switch recombination (CSR). HSM and CSR are unlinked, but share initiating and mechanistic factors. Prior evidence indicates CLL B cells are antigen experienced; however, half of all CLL patients have an unmutated IGHV and follow more aggressive clinical courses. The biologic importance of the CLL immunoglobulin isotype and CSR in B cell receptor (BCR)-mediated signaling is not well understood. We hypothesized the immunoglobulin isotype and expression density of surface immunoglobulin would correlate with IGHV mutation status and other clinical parameters, and predict BCR responsiveness in vitro. Methods: 195 samples from our CLL specimen repository with detailed clinical and biologic characterization were evaluated. Surface expression of IgD, IgM and IgG was determined using multi-parameter flow cytometry and samples were classified as either IgG+ or IgM+IgD+ co-expressing. An ROC analysis was performed to identify the most discriminate value for dichotomization of IgM surface expression as a function of IGHV mutation status. IgMhiIgD+, and IgMlowIgD+ patients were then analyzed for associations with clinical and biologic parameters. To investigate the biological mechanisms of potential associations, BCR-mediated signaling after isotype specific crosslinking in vitro was measured by quantification of downstream phospho-proteins (SYK, AKT, ERK, MEK1, NFκB) by flow cytometry (phospho-flow). Samples were crosslinked with IgM (or IgG if IgG+), IgD, and isotype control and incubated for 0, 15, 30, 60, and 120 minutes. The extent of phosphorylation at each timepoint was expressed as mean fluorointensity (MFI) of the examined proteins. Crosslinked samples were also analyzed using oligonucleotide microarray gene analysis (GEP) to assess differential transcription. Results: A correlation was identified between the percentage of CLL cells expressing surface IgM and IGHV mutation status (r2=0.33); higher surface expression of IgM correlated with unmutated IGHV. The most discriminate value of IgM expression for prediction of IGHV mutation was 47%, with an area under ROC curve of 0.72. Of the 195 patient samples analyzed, 91 (47%) were IgMhiIgD+; 85 (43%) were IgMlowIgD+; and 19 (10%) were IgG+. IgMhiIgD+ samples showed increased expression of CD38 (p<0.01) and ZAP70 (p<0.05) compared to IgMlowIgD+ samples. Only 1 of 19 IgG+ samples was IGHV unmutated. Neither immunoglobulin isotype nor density correlated with gender, lymphocyte doubling time, age, or Rai stage at diagnosis. There was a trend toward shorter time to first treatment by IgM expression (IgMhiIgD+ 7.9yrs vs. IgMlowIgD+ 9.4yrs) but this was not significant (p=0.09). To date, we have tested the biologic relevance of surface immunoglobulin (sIg) density and isotype in 13 patients using phospho-flow analysis. Crosslinking in the IgMlow patients (sIgM <30%; n=5) demonstrated no change in MFI of tested proteins. Among IgMmod patients (sIg 31–60%; n=2), a maximal 3 fold increase in p-SYK MFI with IgM crosslinking and 3.8 fold increase with IgD crosslinking was observed. In IgMhi samples (sIgM >60%; n=3), there was a maximal 3.5 fold increase in MFI with IgM but no change with IgD crosslinking. In the IgG+ samples (n=3), a maximum 5 fold increase in MFI was observed. Analysis of GEP data is ongoing. Conclusion: Surface expression of IgM in CLL varies considerably between patients. This difference in density of surface IgM expression correlates with markers of clinical risk. Although we do not propose immunoglobulin isotype or expression density as a novel prognostic factor, the findings that high surface expression of IgM in CLL predicts BCR mediated signaling after crosslinking provides insight into CLL immunobiology and disease responsiveness to antigens in the microenvironment. In addition, the observation of significant BCR activation within the IgG+ population (which was predominantly IGHV mutated) suggests that the responsiveness of the BCR to crosslinking may be related to the immunoglobulin isotype and density, and not solely determined by the IGHV mutation status. Disclosures: No relevant conflicts of interest to declare.

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FRI0015 PHENOTYPE AND FUNCTION OF THE PERIPHERAL BLOOD DENDRITIC CELLS OF PSORIASIS PATIENTS WITH AND WITHOUT ARTHRITIS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.4296 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 578.1-579

Author(s):

S. Schnitte ◽

A. Fuchs ◽

T. Funk ◽

A. C. Pecher ◽

D. Dörfel ◽

...

Keyword(s):

Risk Factors ◽

Flow Cytometry ◽

Dendritic Cells ◽

Psoriatic Arthritis ◽

T Lymphocytes ◽

Peripheral Blood ◽

Surface Proteins ◽

Research Support ◽

Cross Sectional ◽

Cell Counts

Background:Psoriasis is a frequent skin disease that can appear with an arthritic manifestation in approximately 30% of the cases [1]. The underlying excessive immune reaction caused by pro-inflammatory cytokines can be triggered by several risk factors [2]. Various subgroups of Dendritic cells (DCs) in the skin play a crucial role in the induction of the dermal inflammatory response [3].Objectives:As the role of peripheral blood DCs remains unknown and the cause of an arthritic manifestation is still not completely understood [4], this project aimed to detect differences in phenotype or function of peripheral blood DCs in psoriatic patients with or without arthritis.Methods:We analyzed peripheral blood cells of 60 psoriasis patients with and without arthritis. Different DC subpopulations were detected by flow cytometry. Monocyte-derived DCs were cultured with or without Lipopolysaccharides to gain immature (iDC) and mature (mDC) cells. The DC phenotype was determined by staining with CD80, CD83, CD86, CD206, CCR7, CD1a, HLA-DR, CD40, GPN-MB, DC209 and CD14. Their T-cell stimulatory capability was analyzed by co-incubation with Carboxyfluorescein succinimidyl ester stained lymphocytes and the quantification of CD4+ T-lymphocytes afterwards. To measure the migration capacity DCs were seated into transwell chambers with a semipermeable membrane and partly supplemented with Macrophage Inflammatory Protein 3 Beta (Mip3b). Migrated cells were detected by flow cytometry. Measured cell counts were normalized to cell counts without Mip3b stimulation.Results:Comparing the factor of increase of migrated mDC counts due to mip3b stimulation, we detected a significant lower rate in samples of patients with arthritis (PsA) compared to those of patients without (Ps). Assays of mDCs without mip3b stimulation showed a significant higher count of migrated cells in the samples of the arthritic group [Figure 1]. Cell counts with Mip3b stimulation did vary slightly in the groups. The DC subpopulations and the expression of analyzed cell surface proteins did not show significant differences. The amounts of stimulated T-Lymphocytes did not differ significantly.Figure 1.Migration essay showing mDCs following Mip3b (+miß3b) as multiples of mDCs without stimulation (-mip3b). The factor of increase is significantly lower in patients with arthritis (PsA) compared to patients without (Ps). Absolute counts of migrated mDCs without Mip3b are significantly higher in the arthritic group. Cell counts with stimulation do not differ significantly (data not shown). N=24, p<0.05Conclusion:CCL19 (Mip3b) is a potent ligand to the CCR7 receptor inducing migration of DCs towards the lymphatic node [5]. The CCR7 amounts on the DC surface did not differ significantly in the groups. The mDCs without CCL19 stimulation migrated in higher amounts in samples of arthritic patients. Cell counts of stimulated DCs showed only slight differences. These results could be generated by a different appearance of the DCs of arthritic patients that might facilitate migration. Further experiments focusing on this aspect should be performed. A possible effect of disruptive factors (age, sex, medication…) needs to be clarified.References:[1]Henes, J.C., et al.,High prevalence of psoriatic arthritis in dermatological patients with psoriasis: a cross-sectional study.Rheumatol Int, 2014.34(2): p. 227-34.[2]Lee, E.B., et al.,Psoriasis risk factors and triggers.Cutis, 2018.102(5s): p. 18-20.[3]Kim, T.G., S.H. Kim, and M.G. Lee,The Origin of Skin Dendritic Cell Network and Its Role in Psoriasis.Int J Mol Sci, 2017.19(1).[4]Veale, D.J. and U. Fearon,The pathogenesis of psoriatic arthritis.Lancet, 2018.391(10136): p. 2273-2284.[5]Ricart, B.G., et al.,Dendritic cells distinguish individual chemokine signals through CCR7 and CXCR4.J Immunol, 2011.186(1): p. 53-61.Acknowledgments:This project was financially supported by Novartis Pharma GmbH.Disclosure of Interests:Sarah Schnitte Grant/research support from: Reaserch grant by Novartis, Alexander Fuchs: None declared, Tanja Funk: None declared, Ann-Christin Pecher: None declared, Daniela Dörfel: None declared, Jörg Henes Grant/research support from: Novartis, Roche-Chugai, Consultant of: Novartis, Roche, Celgene, Pfizer, Abbvie, Sanofi, Boehringer-Ingelheim,

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Dissecting Macrophage-Derived Microvesicles' Content and Function.

Blood ◽

10.1182/blood.v116.21.3785.3785 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3785-3785

Author(s):

Noura Ismail ◽

Kara Batte ◽

Leni Moldovan ◽

Clay Marsh ◽

Melissa Piper

Keyword(s):

Flow Cytometry ◽

Surface Antigen ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Antigen Expression ◽

Mononuclear Phagocytes ◽

Gm Csf ◽

Wide Range ◽

Surface Antigen Expression ◽

And Function

Abstract Abstract 3785 Microvesicles (MVs) are small membrane-bound vesicles released under normal homeostatic and stimulatory conditions by a wide variety of cell types. Microvesicles are collectively referred to as exosomes and microparticles which vary in size due to different cellular mechanisms responsible for their production. These microvesicles have a wide range of functions from facilitating communication to regulating cellular growth and differentiation. During their production; microvesicles become enriched in various molecules including proteins and nucleic acids. Previously, we have shown that plasma microvesicles derived from many cell lineages contain microRNAs (miRNAs). We also found that the majority of the peripheral blood microvesicles are derived from platelets while those originating from monocytic cells including macrophages represent the second largest population. Since microvesicles derived from mononuclear phagocytes are a large subpopulation in the plasma; we were interested in understanding their content and function. We hypothesized that the content and/or quantity of macrophage-derived microvesicles could induce the maturation of monocytes. To address our hypothesis, peripheral blood monocytes were treated in vitro for 4hr with GM-CSF; washed and cultured in media devoid of cytokines for 24 h then microvesicles were collected. Flow cytometry and electron)confocal microscropy were used to quantify and visualize microvesicles production. To examine the function of the microvesicles on macrophage maturation, the purified microvesicles were then cultured with freshly isolated monocytes. Macrophage differentiation was determined by cellular adherence using a crystal violet uptake assay and changes in surface antigen expression by flow cytometry. We also examined the genetic changes induced in monocytes incubated with the microvesicles compared to GM-CSF-treated cells. We found that freshly isolated monocytes treated with microvesicles from macrophages acquired phenotypic characteristics of a macrophage such as cellular adherence and surface antigen expression. We also found that treatment of naïve monocytes with the microvesicles induced molecular changes similar to GM-CSF treated monocytes. We found more than 7985 mRNAs that were similarly expressed between the two culture conditions. Notably, we observed the unique expression of 1324 and 1079 genes in the GM-CSF-treated compared to the microvesicle-treated cells, respectively. To begin dissecting the molecules contained in the microvesicles responsible for these changes, we performed mass-spectrometry and miRNA profiling. We observed the expression of miRs-223, -222,-191, -484, -016, -026a, and -155 in GM-CSF-derived microvesicles. Notably, these miRNAs were also expressed in the cells from which the microvesicles were released. We have begun bioinformatics analyses to predict whether the expression of the miRNAs may account for the decrease expression of specific genes in cells treated with the microvesicles that undergo differentiation. Many of the proteins found in the vesicles are important in facilitating protein:protein interactions and nucleic acid binding. Based on our observations; we postulate that microvesicles in areas of inflammation may contribute to the inflammatory response through the maturation of immune cells and activation of cells responsible for tissue repair. Thus, defining key components of this response may identify targets to regulate inflammation. Disclosures: No relevant conflicts of interest to declare.

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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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Increased Platelet-Derived CD40L May Modulate Endothelial Cell ICAM-1 Expression and Interact With Leukocytes In Sickle Cell Anemia

Blood ◽

10.1182/blood.v122.21.974.974 ◽

2013 ◽

Vol 122 (21) ◽

pp. 974-974

Author(s):

Vanessa Tonin Garrido ◽

Renata Proença-Ferreira ◽

Venina M. Dominical ◽

Marcos André Cavalcanti Bezerra ◽

Aderson S. Araujo ◽

...

Keyword(s):

Flow Cytometry ◽

Endothelial Cells ◽

Steady State ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Repeated Measures ◽

Conflicts Of Interest ◽

Endothelial Activation ◽

Surface Expression ◽

Platelet Surface

Abstract Background Vaso-occlusive events are a major cause of morbidity in sickle cell anemia (SCA) and attributable to the abnormal adhesion of red cells and leukocytes to the endothelium. Platelets may contribute to the chronic inflammation and endothelial activation that initiates the vaso-occlusive process. We hypothesized that platelet-associated CD40 ligand (CD40L) may contribute to platelet-mediated inflammatory responses in SCA. Aims This study evaluated the platelet (PLT) release of CD40L, the expression of its receptor (CD40) on platelets, neutrophils, lymphocytes and monocytes of control individuals (CON) and SCA patients, and also the ability of platelet-derived CD40L to activate endothelial cells. Methods IL-8, soluble ICAM-1, VCAM-1 and CD40L were determined in PLT-free plasma or the supernatant of stimulated (ADP or Collagen) and unstimulated PLTs (2•10⁸/mL in Kreb’s buffer), from CON individuals and steady-state SCA patients, by ELISA. Flow cytometry was used to analyze CD40 expression on platelets, neutrophils, lymphocytes and monocytes from the peripheral blood of the study’s subjects. Human umbilical vein endothelial cells (HUVECs) were cultured (1x106cells/well; 37°C, 5% CO2) together with PLTs (3x108PLTs/well) from CON individuals or steady-state SCA patients for 24h, 37°C, 5%CO2, in the presence, or not, of blocking antibodies against CD40L. After incubation, PLTs were removed and HUVECs analyzed by flow cytometry for CD54 (ICAM-1) surface expression. Results SCA individuals presented elevated levels of plasma CD40L (724.4± 55.7 pg/ml; n=90) compared to CON (241.5±34.6 pg/ml; n=41; P<0.0001) and these levels correlated with PLT counts (rs=0.255; P=0.015). No correlation was found between plasma CD40L and plasma IL-8, ICAM-1 or VCAM-1. PLT release of CD40L (90 min, 37°C, 5%CO2) was evaluated; PLTs of SCA patients released higher quantities of CD40L (8347±1464 pg/108 PLTs; n=10) than PLTs of CON individuals (3652±568 pg/108 PLTs; n=5; P=0.019). CD40L release from SCA PLTs was augmented by incubation with collagen (P<0.001), but not ADP. Expression of the CD40 receptor on the platelet surface was elevated in the SCA group (52.4±2.7% positive cells; n=23), compared to the CON group (36.8±3.7% positive cells; n=9; P=0.005). The surface expression of CD40 was also elevated on neutrophils (SCA, 10.4±1.5% positive cells, n=14; CON, 5.5±1.1% positive cells, n=13; P=0.03), lymphocytes (SCA, 8.3±0.8% positive cells, n=16; CON, 3.6±0.4% positive cells, n=14; P<0.001) and monocytes (SCA 69.6±5.9% positive cells, n=16; CON, 49.9±5.8% positive cells, n=14; P=0.03) of SCA patients, compared to controls. ICAM-1 expression on the surface of HUVECs (Basal expression 32.8±1.8%, n=11) was significantly increased following incubation with SCA PLTs (54.0±4.8%, n=11, p<0.0001) and slightly augmented after incubation with CON PLTs (40.8±3.1%, n=11, p<0.05; Repeated measures ANOVA). Interestingly, when HUVECs and SCA PLTs were incubated with a blocking antibody against CD40L, the increase in ICAM-1 expression was significantly reversed on HUVECs (HUVECs, 28.1±0.2%, n=6; HUVECs+SCA PLTs, 42.0±3.3%, n=6; HUVECs+SCA PLTs+anti-CD40L 28.9±1.5%, n=6; P<0.01). Conclusions Plasma levels and platelet release of CD40L were found to be significantly elevated in SCA, in association with increased expressions of the CD40 receptor on SCA PLTs, neutrophils, lymphocytes and monocytes, possibly indicating a CD40L-mediated crosstalk between platelets and leukocytes in SCA. Platelets from SCA patients can induce adhesion molecule expression on the surface of endothelial cells in vitro, and this up-regulation may be modulated by platelet-derived CD40L. Results suggest that the CD40/CD40L pathway may be altered in SCA and that platelets may participate in this up-regulation. Given the potent inflammatory effect of this cytokine, a role for platelets and this cytokine in endothelial activation, inflammation and consequent vaso-occlusion, is likely. Disclosures: No relevant conflicts of interest to declare.

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Rapid Prediction of Peripheral Blood CD34 Counts Using a Multiple Regression Model Derived from Automated Hematology Analyzer Cell Population Data

Blood ◽

10.1182/blood.v126.23.3099.3099 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3099-3099 ◽

Cited By ~ 1

Author(s):

Thomas Porturas ◽

Mary Sell ◽

Leah Irwin ◽

Una O'Doherty ◽

Carlos Hipolito Villa

Keyword(s):

Flow Cytometry ◽

Stem Cell ◽

Multiple Regression ◽

Peripheral Blood ◽

Population Data ◽

Blood Samples ◽

Peripheral Blood Cd34 ◽

Cell Counts ◽

Flow Cytometric ◽

Hematology Analyzer

Abstract Background: Although peripheral blood CD34+ stem cell counts by flow cytometry correlate well with yields, the time, complexity, and cost associated with flow cytometry limits its utility. Rapid, cost-effective, surrogate predictors (with <1hr turnaround) would allow for same-visit analyses and alteration of collection and mobilization strategies, particularly for the optimal use of time-sensitive and costly agents such as plerixafor. We previously demonstrated that morphologic parameters of neutrophil-like cells measured by hematology analyzers correlated with CD34 counts. We aimed to improve these models by using multiple regression analyses on data from a common hematology analyzer. Methods: Patients undergoing stem cell apheresis were evaluated over a 6 month period. The day prior to initiation of apheresis, and on the morning of initial collection, peripheral blood samples were drawn into EDTA collection tubes and flow cytometric CD34 measurement and/or CBCs were performed on the Beckman Coulter DxH 800 hematology analyzer per standard protocol. CD34 cells were counted by flow cytometric ISHAGE protocols. Data from the DxH (48 variables per specimen) were exported into a data matrix with the corresponding flow cytometric data. Multiple regression analysis was performed using a step-wise method with log(peripheral CD34) as the dependent variable (SPSS, IBM). Data were randomly selected into a training-set of 70% of cases and a test-set of 30% of cases for validation. The derived model was further tested against peripheral blood data from the morning of collection to predict harvest yields. Further analyses were performed using Prism (GraphPad). Results: Tandem peripheral blood CD34 counts and CBC cell-population data were obtained from 69 blood samples in 64 patients. The population included patients with multiple myeloma (45), non-Hodgkin lymphoma (12), Hodgkin lymphoma (5), and amyloidosis (2). 41% of patients were female. In the test data set examining collection yields, 37 patients were mobilized with GCSF (+/- chemotherapy) alone, while 17 had plerixafor added to the regimen. 33 of these patients had same-day CBC data available for model prediction. The median processed volume was 15 L (range 5.9 to 19.7). The model to predict peripheral CD34 counts incorporated 3 variables from the hematology analyzer data (SD-V-EGC, SD-C-EGC, and NE#). Interestingly, the model included two variables descriptive of the morphology of early granulocytic cells. The model demonstrated an R value of 0.829 (adjusted R2 = 0.670, figure 1a). In testing the morning-of-collection model-predicted peripheral CD34, we found the model performed similarly to flow cytometry in predicting 1st collection yields. Furthermore, the CD34 prediction using the model (Figure 1 b) resulted in similar correlation with first-collection yields in patients treated with plerixafor versus patients not treated with plerixafor, in contrast to day-prior CD34 counts by flow-cytometry (Figure 1c). Two outliers for CD34 cell yield based on model predicted peripheral CD34 were identified. In one patient, the processed volume was very low (6.8 L, <5% percentile), while the second had a low mononuclear cell collection efficiency (35%) compared to the mean in this population (58.7%±23.3%). Threshold values for the model accurately identified patients appropriate for collection initiation (or plerixafor administration). Conclusion: Using data from a common, automated CBC analyzer, we developed a rapid, less-costly, and simple model to predict CD34 cell counts and 1st harvest yields. Because the measurement results can be obtained within the same clinic visit, and can be repeated with each CBC, the model is particularly useful to guide optimal use of plerixafor. We also envision that the model is useful for quality assurance of collection by identifying patients in whom cell yields were sub-optimal with respect to predicted CD34 cell counts. Additional studies to test the model in a larger population are ongoing. We propose that this model (and similarly derived models) can be implemented in clinical planning algorithms to improve the efficiency and cost of stem cell collection by apheresis. Acknowledgments: We would like to acknowledge and the nurses and staff of the apheresis unit and the stem cell and flow cytometry laboratories at the Hospital of the University of Pennsylvania for their contributions. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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The Morphology and Cellular Characterization of Thrombocytes in Adult Xenopus laevis.

Blood ◽

10.1182/blood.v106.11.3947.3947 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3947-3947

Author(s):

Takako Ishida ◽

Miyako Obuchi-Shimoji ◽

Takeshi Kuribara ◽

Nami Nogawa ◽

Tomoyuki Tahara ◽

...

Keyword(s):

Flow Cytometry ◽

Xenopus Laevis ◽

Peripheral Blood ◽

Blood Cells ◽

Structural Characteristics ◽

Flow Cytometric Analysis ◽

Peripheral Blood Cells ◽

Morphological Aspect ◽

Cell Counts

Abstract In primates and rodents, platelets originate from the bone marrow megakaryocytes through a unique differentiation process with nuclear polyploidization, cytoplasmic maturation and proplatelet formation. In contrast, circulating thrombocytes of most non-mammalian vertebrates are particularly distinctive; the cells are large and nucleated. Adult Xenopus laevis may be an useful non-mammalian model for analyzing dynamic hematopoiesis because they are individually tolerable for time lapse analysis in vivo with sequential blood sampling, whereas classification of cell types has not been established yet. Microstructures of Xenopus thrombocytes observed with electron microscope exhibited structural characteristics largely resembling zebrafish thrombocytes with nucleated spindle cellular features (Thattaliyath et al., Blood 2005), and they had lobulated nuclear chromatin, granules, microparticles and open canalicular system-like-structures as in mammalian megakaryocytes. Since thrombocyte identification based on the morphological aspect was not sufficient, chemical staining with acetylecholinesterase and thiazole orange were performed. Additionally, mice were immunized by Xenopus peripheral blood cells to generate monoclonal antibodies, and two hybridomas producing IgG, respectively T12 and T5, were screened. T12+ (T12 positive) cells were morphologically typical thrombocytes. Flow cytometric analysis revealed that T12+ cells were also positive to anti-human GpIIb/IIIa polyclonal antibodies, and approximately 2-3% of whole peripheral blood cells were T12+/GpIIb/IIIa+ that distributed in FSClow/SSClow fraction. When T12 was injected into Xenopus to deplete T12+ cells in vivo, the detectable level of T12 in the circulation lasted for more than several weeks. Peripheral thrombocyte counts predominantly began to decrease immediately and reached their nadir at day 3, but white blood cell counts were not changed. RNA-rich blood cells considered as younger cells were then increasingly appeared, and finally the cell counts recovered to normal levels at day 10–15, indicating that in vivo depletion of T12+ cells induced thrombopoiesis and/or release of mature thrombocytes from the pool. T5 recognizing cells were classified into two populations by immunostaining and flow cytometry; T5+/GpIIb/IIIa+ cells were morphologically thrombocytic as the cells recognized by T12, while T5+/GpIIb/IIIa− cells were spherical and similar appearance to lymphocytic cells. These observations raised some possibilities e.g.; antigen of T5 was a membrane protein common to both lymphocytes and thrombocytes, or T5+/GpIIb/IIIa− cells were thrombocyte progenitors at earlier development stage than T12+/GpIIb/IIIa+ cells. Nevertheless only a few percent of T12+ and T5+ cells resided in peripheral blood, immunostaining revealed that the proportions of T12+/T5+ and T5+ cells in spleen were 10% and 70%, and T12+/T5+ and T5+ cells in liver were 5% and 20%, respectively. These suggest that spleen is predominantly involved in thrombopoiesis and/or thrombocyte storage in adult Xenopus. As T12 and T5 can be used successfully in flow cytometry and magnetic cell sorting, they should contribute us directly to elucidate the origin of circulating Xenopus thrombocytes and their cellular development process.

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Prevalence of 46/1 JAK2 Haplotype in Patients with Budd-Chiari Syndrome with and without JAK2V617F and TET2 Mutations.

Blood ◽

10.1182/blood.v114.22.434.434 ◽

2009 ◽

Vol 114 (22) ◽

pp. 434-434

Author(s):

Nicholas C Lea ◽

Lara N Roberts ◽

Raj K Patel ◽

Rachel Westbrook ◽

Michael A Heneghan ◽

...

Keyword(s):

Bone Marrow ◽

Skin Biopsy ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Family Members ◽

Uniparental Disomy ◽

Cellular Growth ◽

Jak2v617f Mutation ◽

Budd Chiari Syndrome ◽

Chiari Syndrome

Abstract Abstract 434 Budd-Chiari Syndrome (BCS) is a group of disorders resulting from obstruction to hepatic venous outflow; myeloproliferative disorder (MPD) accounts for 10-40% of cases. We previously described latent MPD in 58.5% of patients with idiopathic BCS, detected with allele-specific PCR for the JAK2V617F mutation and proposed its use as a screening tool for occult MPD. A predisposing germline JAK2 haplotype (designated 46/1) has since been described as a strong genetic risk factor for MPD and may further characterise latent MPD in BCS. We studied 28 patients with BCS (23 from our original cohort; female n=16, mean age 30.3 years, SD 10) presenting between 1985 and 2008; 14 with the JAK2V617F mutation. Genomic DNA was obtained from archived bone marrow films, fractionated and unfractionated peripheral blood or bone marrow leucocytes. Skin biopsy or CD3+ cells were used as a source of constitutional DNA. DNA was analysed by pyrosequencing for 2 SNPs (rs12340895, rs12343867) which tag the 46/1 JAK2 haplotype. The 46/1 haplotype was detected in 16/28 (57.1%) subjects; 50% of those with the JAK2V617F mutation and 64.3% of those without it. The prevalence in those lacking the JAK2V617F mutation is significantly higher than the frequency in the Wellcome Trust Case Control Consortium cohort of 24% (P=0.0023). 3/28 subjects had previously diagnosed JAK2V617F positive Polycythemia Vera (PV) and all had the 46/1 haplotype, resulting in a prevalence of 36.4% in those with JAK2V617F positive latent MPD. Age at presentation of BCS was significantly lower in those with the 46/1 haplotype (26.4 years compared to 34.8 years, P=0.03). This difference remained significant in those lacking the JAK2V617F mutation (24.0 years compared to 37.6 years, P=0.024) but was not seen in those with the JAK2V617F mutation (P=0.547). There was no difference in presenting clinical features, haematological parameters or treatment between those with and without the 46/1 haplotype. Overall survival in 26/28 patients was 76.9% (median 90 months, range 2 days to 266 months). 17/28 subjects underwent OLT of which 14/17 (11/12 with 46/1 haplotype) are alive at a median of 90 months post transplant (range 9-266 months). 3/17 patients developed post-OLT veno-occlusive disease, all with the JAK2V617F mutation and 2/3 with the 46/1 haplotype. Overt MPD has not developed in any patient without the JAK2V617F mutation; repeat JAK2 mutational analysis was undertaken in 3/14 (2/3 with 46/1 haplotype) and none have acquired the mutation at a mean of 54 months. 19/28 cases were genotyped using SNP markers (Affymetrix SNP6); 3/19 have acquired uniparental disomy (aUPD) on 9p overlapping the JAK2 gene. As TET2 has been postulated as a ‘pre-JAK2' aberration, we sequenced the complete TET2 gene using massively parallel high throughput sequencing (Roche 454); 2/15 patients samples were positive for TET2 mutations. One of our cases had a familial history of PV; the patient, his father and uncle all have JAK2V617F positive PV and were heterozygous for the 46/1 haplotype in DNA extracted from a skin biopsy. 2/3 were homozygous for both the 46/1 haplotype and JAK2V617F mutation in bone marrow granulocytes with SNP6 array data confirming aUPD on 9p. JAK2V617F was detected in cultured in vitro colonies from all 3 family members. All 3 affected family members had normal cytogenetics and normal TET2 gene. 3 unaffected siblings were heterozygous for the 46/1 haplotype both in peripheral blood, CD3+ cells and granulocytes but negative for JAK2V617F mutation and lacked aUPD on 9p. We have found a highly significant prevalence of the 46/1 haplotype in our cohort of BCS, as well as in family members of a patient with JAK2V617F positive BCS and PV. The 46/1 haplotype was detected in patients with idiopathic BCS with and without the JAK2V617F mutation, suggesting a predisposition to idiopathic BCS independent of JAK2V617F mutation acquisition and latent MPD. The prevalence and lower age of presentation in those with the 46/1 haplotype lacking the JAK2V617F mutation supports an alternate, as yet unknown, mechanism predisposing to BCS. The presence of the 46/1 haplotype in unaffected relatives of our JAK2V617F BCS patient suggests that additional germline variation may predispose to or protect from acquisition of JAK2V617F positive disease. Alternatively the 46/1 haplotype may directly confer a cellular growth advantage via increased responsiveness of JAK2 to cytokine stimulation. 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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