Light Scatter Properties Research Articles

The Prognostic Significance of Polyclonal Bone Marrow Plasma Cells in Patients with Actively Relapsing Multiple Myeloma

Background: Prior studies suggest that the presence of >5% polyclonal plasma cells (pPCs) among total plasma cells (PCs) within the bone marrow (BM) is associated with a longer progression-free survival, higher response rates, and lower frequency of high-risk cytogenetic abnormalities in patients with newly diagnosed multiple myeloma (MM). However, the incidence and prognostic utility of this factor in patients with relapsed and/or refractory MM has not been previously evaluated. Thus, we evaluated the prognostic value of quantifying the percentage of pPCs among the total PCs in the BM of patients with actively relapsing MM.Methods: We evaluated all MM patients with actively relapsing disease (biochemical and/or symptomatic) seen at the Mayo Clinic, Rochester, from 2012 to 2013, who had BM samples evaluated by seven-color multiparametric flow cytometry. All patients had at least 24 months of follow-up from the date of flow evaluation. Cell surface antigens were assessed by direct immunofluorescence antibodies for CD45, CD19, CD38, CD138, cytoplasmic Kappa and Lambda Ig light chains, and DAPI nuclear stain. The flow cytometry data was collected using the Becton Dickinson FACSCanto II instruments that analyzed 150,000 events (cells); this data was then analyzed by multi-parameter analysis using the BD FACS DIVA Software. PCs were selectively analyzed through combinatorial gating using light scatter properties and CD38, CD138, CD19, and CD45. Clonal PCs were separated from pPCs based on the differential expression of CD45, CD19, DAPI (in non-diploid cases), and immunoglobulin light chains. The percentage of pPCs was calculated in total PCs detected. Survival analysis was performed by the Kaplan-Meier method and differences were assessed using the log rank test.Results: There were 180 consecutive patients with actively relapsing MM who had BM biopsies analyzed via flow cytometry as part of their routine clinical evaluation. The median age of this group was 65 years (range: 40 - 87); 52% were male. At the time of this analysis, 104 patients had died, and the 2-year overall survival (OS) rate for the cohort was 58%. The median number of therapies received was 4 (range: 1 - 15). Of these patients, 61% received a prior ASCT, and almost all (99%) received prior regimens containing either immunomodulators or proteasome inhibitors. There were 55 (30%) patients with >5% pPCs among the total PCs in their BM. The median percentage of pPCs among total PCs in these 55 patients was 33% (range: 5 - 99). The median OS for those with >5% pPCs was not reached compared with 22 months for those with <5% pPCs (P = 0.028; Figure 1). Patients with <5% pPCs PCs had a higher likelihood of high-risk FISH cytogenetics compared with the rest of the patients. In a univariate analysis, increasing number of pPCs was associated with an improved OS, while higher labeling index, number of prior therapies, and the presence of high-risk FISH cytogenetics were associated with a worse OS. In a multivariate analysis, only the increasing number of pPCs (P = 0.006), higher labeling index (P = 0.0002) and number of prior therapies (P = 0.003) retained statistical significance.Conclusion: Quantitative estimation of the percentage of pPCs among the total PCs in the BM of patients with actively relapsing MM was determined to be a predictor of worse OS. As such, this parameter is able to identify a group of patients with MM with actively relapsing disease who have a particularly poor outcome. Further studies evaluating its biological significance are warranted. [Display omitted] DisclosuresKapoor:Celgene: Research Funding; Amgen: Research Funding; Takeda: Research Funding. Gertz:Prothena Therapeutics: Research Funding; Novartis: Research Funding; Alnylam Pharmaceuticals: Research Funding; Research to Practice: Honoraria, Speakers Bureau; Med Learning Group: Honoraria, Speakers Bureau; Celgene: Honoraria; NCI Frederick: Honoraria; Sandoz Inc: Honoraria; GSK: Honoraria; Ionis: Research Funding; Annexon Biosciences: Research Funding. Kumar:AbbVie: Research Funding; Noxxon Pharma: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding; Sanofi: Consultancy, Research Funding; Onyx: Consultancy, Research Funding; Skyline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Research Funding; Kesios: Consultancy; Glycomimetics: Consultancy; BMS: Consultancy.

Cell size assays for mass cytometry.

Mass cytometry offers the advantage of allowing the simultaneous measurement of a greater number parameters than conventional flow cytometry. However, to date, mass cytometry has lacked a reliable alternative to the light scatter properties that are commonly used as a cell size metric in flow cytometry (forward scatter intensity-FSC). Here, we report the development of two plasma membrane staining assays to evaluate mammalian cell size in mass cytometry experiments. One is based on wheat germ agglutinin (WGA) staining and the other on Osmium tetroxide (OsO4 ) staining, both of which have preferential affinity for cell membranes. We first perform imaging and flow cytometry experiments to establish a relationship between WGA staining intensity and traditional measures of cell size. We then incorporate WGA staining in mass cytometry analysis of human whole blood and show that WGA staining intensity has reproducible patterns within and across immune cell subsets that have distinct cell sizes. Lastly, we stain PBMCs or dissociated lung tissue with both WGA and OsO4 ; mass cytometry analysis demonstrates that the two staining intensities correlate well with one another. We conclude that both WGA and OsO4 may be used to acquire cell size-related parameters in mass cytometry experiments, and expect these stains to be broadly useful in expanding the range of parameters that can be measured in mass cytometry experiments. © 2016 International Society for Advancement of Cytometry.

Abstract 5082: A quantitative and multi-parameter flow cytometry assay to simultaneously assess cell migration and immunophenotype

Abstract Cell migration occurs during physiological and pathological processes, which include wound healing and tumor metastasis. In vitro migration assays are necessary to understand the mechanism underlying cell migration and also to identify inhibitory or stimulatory molecules. The Boyden chamber assay is commonly used to measure cell motility in vitro where the number of cells that migrate through the transwell membrane is quantitated manually using an inverted microscope. Alternatively, cells can be detached from the membrane and stained with a fluorescent probe, prior to counting cells using a plate reader. These conventional assays are limited by an inability to simultaneously characterize individual cells while monitoring migration. To address this, we have developed a multi-parameter flow cytometry-based migration assay for the quantitative measurement of cell migration and simultaneous immunophenotypic analysis of the migrated cells. Invasive HT-1080 and non-invasive MCF-7 cancer cell lines were plated in the upper layer of a cell permeable membrane, as per the standard Boyden chamber assay. BD Horizon™ Fixable Viability Stain (FVS) was used to determine the optimal detachment conditions providing maximum yield with minimal impact on cell viability. Migrated cells were also co-stained with multiple conjugated antibodies to assess cell surface marker expression. Cells were analyzed on BD Accuri™ flow cytometer for rapid quantitation of migrated cells and analysis of up to 4 parameters. Alternatively, BD FACSCelesta™ flow cytometer was used for higher parameter analysis. Migrated cells were identified based on light scatter properties and viability staining with FVS, while immunophenotype was assessed in live cells. Our results demonstrate that this flow cytometric assay can be used to rapidly quantify changes in the migratory ability of different cell lines in the presence of inhibitors or stimulators. Additionally, we were able to assess the expression of hallmark cancer cell markers (CD44, CD24, CD326 (EpCAM)) before and after migration. The ability to quantify the number of migrating cells in response to specific stimuli and to simultaneously define cell immunophenotype represents a significant advancement, as compared to conventional cell motility assays. Our results demonstrate that this novel approach allows for a deeper analysis of cell migration that could enable complex drug discovery studies and the discovery of new cell signatures associated with metastatic progression. Citation Format: Mirko Corselli, Xiao Wang, Lissette Wilensky, Aaron Middlebrook, Smita Ghanekar, Jacob Rabenstein, Nil Emre. A quantitative and multi-parameter flow cytometry assay to simultaneously assess cell migration and immunophenotype. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5082.

Quantification of circulating clonal plasma cells (cPCs) via multiparametric flow cytometry (MFC) to identify patients with smoldering multiple myeloma (SMM) at high risk of progression.

8015Background: The presence of high numbers of cPCs in patients with SMM, detected by a slide-based immunofluorescence assay, has been associated with a shorter time to progression (TTP) to multiple myeloma (MM). The significance of quantifying cPCs via MFC, a much more readily available diagnostic modality, in patients with SMM has not been evaluated. Methods: This study evaluated patients with a known or new diagnosis of SMM who were seen at the Mayo Clinic, Rochester from January 2008 until December 2013 and who had their peripheral blood evaluated for cPCs by six-color MFC. cPCs were selectively analyzed through combinatorial gating using light scatter properties and CD38, CD138, CD19, and CD45. The cPCs detected were reported out as the number of clonal events/150,000 collected total events. Survival analysis was performed by the Kaplan-Meier method and differences assessed using the log rank test. Results: There were 100 patients with a known diagnosis of SMM who had their peripheral blood evaluate...

Optimization of Cell Culture Conditions for the Earthworm Eisenia hortensis: a Study Investigating the Effects of Media, Carbon Dioxide, Temperature, Serum, and Anti-Fungal Agents

ABSTRACT The aim of this study was to determine the optimal conditions for culturing coelomocytes (leukocytelike cells) from the annelid Eisenia hortensis. It was of particular interest to determine if CO2 could be omitted to permit more wide-spread use of earthworms in cell biology curricula using standard incubators. Two different types of media, DMEM and SFX-Insect Media, were used at varying conditions including: temperature, serum concentration, antimycotic concentration, CO2, and time. Cell viability was measured using propidium iodide and flow cytometry in addition to analysis of forward and side light scatter properties. It was found that the coelomocytes of E. hortensis exhibit the highest level of cell viability when cultured with DMEM supplemented with 10% newborn calf serum at 25 °C. Longer incubations showed lower cell death when CO2 was provided, but CO2 could be omitted for shorter periods of culture without significant loss of cell viability providing 10 mM HEPES was included in the culture medium. It was also observed that SFX-Insect Medium was a suitable alternative to DMEM and was used without the need for 5% CO2, but a minimum of 5% serum needed to be included. The toxicity of amphotericin B was tested and 0.875 μg/ml in DMEM and SFX-Insect Medium did not compromise cell viability. This information shows that earthworms can be cultured easily without the need for a CO2 incubator, thus simplifying laboratory conditions and minimizing costs associated with using earthworms for cell biology curricula and research purposes.

Combination of CD157 and FLAER to Detect Peripheral Blood Eosinophils by Multiparameter Flow Cytometry.

The identification of eosinophils by flow cytometry is difficult because most of the surface antigens expressed by eosinophils are shared with neutrophils. Some methods have been proposed, generally based on differential light scatter properties, enhanced autofluorescence, lack of CD16 or selective positivity of CD52. Such methods, however, show several limitations. In the present study we report a novel method based on the analysis of glycosylphosphatidylinositol (GPI)-linked molecules. The combination of CD157 and FLAER was used, since FLAER recognizes all GPI-linked molecules, while CD157 is absent on the membrane of eosinophils and expressed by neutrophils. Peripheral blood samples from normal subjects and patients with variable percentages of eosinophils (n = 31), and without any evidence for circulating immature myeloid cells, were stained with the combination of FLAER-Alexa Fluor and CD157-PE. A FascCanto II cytometer was used. Granulocytes were gated after CD33 staining and eosinophils were identified as CD157(-)/FLAER(+) events. Neutrophils were identified as CD157(+)/FLAER(+) events. The percentages of eosinophils detected by this method showed a very significant correlation both with automated counting and with manual counting (r = 0.981 and 0.989, respectively). Sorting assays were carried out by a S3 Cell Sorter: cytospins obtained from CD157(-)/FLAER(+) events consisted of 100% eosinophils, while samples from CD157(+)/FLAER(+) events were represented only by neutrophils. In conclusion, this method shows high sensitivity and specificity in order to distinguish eosinophils from neutrophils by flow cytometry. However, since CD157 is gradually up-regulated throughout bone marrow myeloid maturation, our method cannot be applied to cases characterized by immature myeloid cells.

OMIP-023: 10-color, 13 antibody panel for in-depth phenotyping of human peripheral blood leukocytes.

THIS panel was developed and optimized to determine the phenotype and activation of 15 different leukocyte subtypes in one run. Leukocytes are identified by expression of CD45 (leu-1) pan leukocyte antigen. Neutrophil (CD16), monocyte (CD14), T(CD3), B-lymphocyte (CD19), and NK-cell (CD16 and CD56) markers are employed. Special gating strategy is used for subtyping of granulocytes (e.g., eosinophils, neutrophils, basophils). For further T-cell phenotyping, CD4/CD8 markers are used for differentiation of four T-cell subtypes, CD25/CD127 for regulatory T cell identification and CD3/CD16/CD56 for NKT-cells, additionally. Special gating strategies have been developed for B-cell, NK-cell, and monocyte subtyping. For detection and analysis of activation also further activation markers (HLA-DR, CD38, CD25, CD127, and CD69) are analyzed. This panel has been established for analysis of human RBC-lysed EDTAtreated whole blood samples and for cord blood (Table 1). Since the starting material is fresh EDTA-treated blood, dead cells are probably not an issue. Thus to save one channel for specific staining the vitality staining was not used in the panel.

Fluorescence as an alternative to light-scatter gating strategies to identify frozen–thawed cells with flow cytometry

Flow cytometry is a key instrument in biological studies, used to identify and analyze cells in suspension. The identification of cells from debris is commonly based on light scatter properties as it has been shown that there is a relationship between forward scattered light and cell volume and this has become common practice in flow cytometry. Cryobiological conditions induce changes in cells that alter their light scatter properties. Cells with membrane damage from freeze–thaw stress produce lower forward scatter signals and may fall below standard forward scatter thresholds. In contrast to light scatter properties that cannot identify damaged cells from debris, fluorescent dyes used in membrane integrity and mitochondrial polarization assays are capable of labeling and discriminating all cells in suspension. Under cryobiological conditions, isolating cell populations is more effectively accomplished by gating on fluorescence rather than light scatter properties. This study shows the limitations of using forward scatter thresholds in flow cytometry to identify and gate cells after exposure to a freeze–thaw protocol and demonstrates the use of fluorescence as an alternative means of identifying and analyzing cells.

Effects of Red Blood Cell Lysing Solutions on the Detection of Peripheral Basophils of Healthy Normals and Sle Patients by Flow Cytometry

To evaluate the effect of four widely used red blood cell lysing solutions on counting and measurement of activation marker of peripheral basophils in normals and systemic lupus erythematosus (SLE) patients by flow cytometry. Our results showed that the light scatter properties including FS and SS value of leukocytes in whole blood were preserved when whole blood samples were lysed in RBC Lysis Buffer and FACS Lysing Solution, while were affected when lysed in distilled water or ACK. By counting basophils, RBC Lysis Buffer and FACS Lysing Solution were almost the same level, while were significantly lower when lysed in distilled water or ACK. The expressions of CD203c on peripheral basophils of SLE patients were significantly higher than those of normals. Comparing the data of CD203c expression obtained demonstrated that there were no significant differences among them, while FACS Lysing Solution treatment leads to a slightly lower staining intensity of CD203c. We provide a solid description that the widely used red blood cell lysing reagents may influence the light scatter properties of leukocytes, the accuracy of quantity of absolute number of the existence of basophil subsets and the quantity of staining intensity of cell-activated marker CD203c fluorescence when measured by flow cytometry.

Prognostic Value Of Quantifying Circulating Plasma Cells By Multiparametric Flow Cytometry In Patients With Relapsed Multiple Myeloma

BackgroundThe presence of circulating plasma cells (PCs) in multiple myeloma (MM) is a known poor prognostic marker. Prior studies have utilized a technically challenging slide-based immunofluorescence technique to detect and quantify the presence of circulating PCs which was not widely adapted in clinical practice. More recently, the routine use of flow cytometry has provided the opportunity to quantitatively assess circulating PCs in MM patients with relative ease. We report the prognostic value of quantifying circulating clonal PCs using multiparametric flow cytometry in MM patients with relapsed disease. MethodsWe evaluated all MM patients seen at the Mayo Clinic, Rochester from 2009 to 2011 with previous or ongoing relapsed disease and who had their peripheral blood samples evaluated by flow cytometry. Each blood sample had its peripheral blood mononuclear cells isolated by ficoll gradient and stained with antibodies to CD45, CD19, CD38, CD138 and cytoplasmic Kappa and Lambda Ig light chains. A six-color multi-parameter flow cytometer (Becton Dickinson FacsCantos II) was used to examine each sample with a target of detecting 150,000 events (cells) that was then analyzed using the Facs Diva Software. Plasma cells were selectively analyzed through combinatorial gating using light scatter properties and CD38, CD138, CD19, and CD45. Normal PC's were then separated from clonal plasma cells based on the differential expression of CD45, CD19 and polytypic Ig light chains. The clonal plasma cells detected were reported out as the number of clonal events/150,000 collected total events. For those samples where less than 150,000 events were gated or examined, the number of final clonal events was adjusted to 150,000 events. Survival analysis was performed by the Kaplan-Meier method and differences in survival assessed using the log rank test. ResultsThere were 647 consecutive patients with a history of treated MM who had a peripheral blood flow cytometry as part of their routine clinical evaluation. The median patient age was 62 years (29-88) and 55% were male. The median time since diagnosis was 12 months (range: 1-363) and the median number of lines of treatment received was 2 (1-11). There were 81 (13%) patients with clonal circulating PCs with a median of 368 cells (4 – 133,464). The 2-year overall survival (OS) for the 81 (13%) patients with any circulating PCs was 17% compared with 65% for those with none (P<0.001). The presence of circulating clonal PCs was associated with high-risk disease by FISH (P <0.001) as well as higher PCLI (P <0.001). We then correlated the presence of circulating clonal PCs with the disease status at flow cytometry assessment. Among the study patients, only 145 (22%) had actively relapsing disease with the remaining 502 (78%) in a plateau including patients in CR. Circulating clonal PCs were more likely to be detected in patients with actively relapsing disease compared with plateau phase (43% vs. 4%, P < 0.001); none of the CR patients and <5% of the remaining plateau phase patients had any detectable circulating clonal PCs.We then restricted the analysis to the actively relapsing patients; the best cutoff predicting 1-year mortality by ROC analysis was 100 events. Based on this, we defined >=100 events as a cutoff for defining the prognostic role of circulating clonal PCs in actively relapsing patients. The median OS for those with >=100 clonal PC events was 12 months compared with not reached for those with <100 events (p<0.001; Figure 1 [Display omitted] ). Among patients with actively relapsing disease, >= 100 circulating PCs was associated with a higher ISS stage, plasma cell labeling index and bone marrow PC% compared with those with <100 circulating PCs. In a multivariable model, only LDH > 222 (HR: 2.08, P = 0.045) and >= 100 circulating PCs (HR: 2.48, P = 0.048) were found to adversely affect OS in actively relapsing patients. ConclusionThe utilization of flow cytometry to quantify circulating clonal PCs in patients with relapsed MM appears to have significant prognostic relevance. In patients with actively relapsing disease, >= 100 circulating PCs predicted for worse OS with a median of 12 months. Future studies are needed to determine if this would allow an opportunity to develop a more risk adapted approach for patients with relapsed disease.▪ Disclosures:Kumar:Celgene: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Onyx: Consultancy, Research Funding. [Display omitted]

Increased Circulating Plasma Cells On Multiparametric Flow Cytometry As An Independent Prognostic Biomarker In Newly Diagnosed Multiple Myeloma: Implications For Redefining High-Risk Myeloma

BackgroundPrior studies have shown that presence of increased circulating plasma cell (PCs) identified using a slide-based immunofluorescence method is an adverse prognostic marker for overall survival in multiple myeloma (MM), and increases the risk of progression in patients with MGUS and smoldering MM. However, utility in clinical settings has been limited by the cumbersome nature of the test and lack of widespread availability. We studied the prognostic value of circulating PCs using sensitive multiparametric flow cytometry that enable us to quantitatively assess circulating PCs in patients with MM. MethodsWe evaluated all newly diagnosed MM patients seen at the Mayo Clinic, Rochester from 2009 to 2011 who had their peripheral blood samples evaluated by flow cytometry prior to therapy. Patients with plasma cell leukemia were excluded. Each blood sample had its peripheral blood mononuclear cells isolated by ficoll gradient and stained with antibodies to CD45, CD19, CD38, CD138 and cytoplasmic Kappa and Lambda Ig light chains. A six-color multi-parameter flow cytometer (Becton Dickinson FacsCantos II) was used to examine each sample with a target of detecting 150,000 events (cells) that was then analyzed using the Facs Diva Software. PCs were selectively analyzed through combinatorial gating using light scatter properties and CD38, CD138, CD19, and CD45. Normal PC's were then separated from clonal PCs based on the differential expression of CD45, CD19 and polytypic Ig light chains. The clonal PCs detected were reported out as the number of clonal events/150,000 collected total events. For those samples where less than 150,000 events were gated or examined, the number of final clonal events was adjusted to 150,000 events. Survival analysis was performed by the Kaplan-Meier method and differences assessed using the log rank test. ResultsThere were 158 consecutive patients with newly diagnosed MM who had their peripheral blood evaluated via flow cytometry as part of their routine clinical evaluation. The median age of this group was 66 years (39-95) and 59% were male. At the time of this analysis, 25 patients had died and the 2-year OS rate for the cohort was 83%. The 2-year OS for the 89 (55%) patients with any circulating PCs was 76% compared with 91% for those with none (P=0.02). The median number of circulating clonal PCs was 33 (range, 0-46,413)/ 150,000 gated events. Using a ROC analysis the best cutoff predicting 1 and 2-year mortality were 435 and 376 events, respectively. Based on this, we defined >=400 events as a cutoff for defining patients with high-risk disease. The median time-to-next-treatment (TTNT) for patients with circulating clonal PCs >=400 (n=37, 23%) was 14 months compared with 26 months for those with <400 events (n=121, 77%) (p<0.001; Fig 1a). The median OS for those with >=400 clonal PC events was 32 months compared with not reached for those with <400 events (p<0.001; Fig 1b). Patients with >= 400 circulating PCs had higher ISS stage, creatinine, LDH, PC labeling index and bone marrow PC% compared with the rest. In a univariate analysis examining the presence of circulating PC >=400, LDH, labeling index, marrow PC% and FISH risk status, only circulating PCs were prognostic for overall survival (P = 0.0011). Among the 89 patients with any circulating PCs, 26 (30%) had CD45+ clonal PCs. The TTNT in patients with a clonal CD45+ population was inferior to those with no CD45+ clonal PCs in circulation (median 11 vs. 19 months, P = 0.034) as was the OS (P = 0.039). [Display omitted] [Display omitted] [Display omitted] ConclusionQuantitative estimation of circulating clonal PCs in patients with newly diagnosed MM is a powerful predictor of early relapse from therapy and mortality. A cutoff of >=400 clonal events/150,000 gated mononuclear events predicts for a median TTNT of 14 months and OS of 32 months. This parameter is more powerful than the current high-risk parameters such as FISH risk status as well as traditional high-risk markers such as ISS stage and LDH levels, and is able to identify a group of patients with particularly poor outcome. Disclosures:Kumar:Celgene: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Onyx: Consultancy, Research Funding.

Personalizing Therapies With Ex Vivo Pharmacological Responses May Uncover The Differences Between IDA-DNR-MIT Among European AML Protocols

Background and objectivesProtocols for acute myeloid leukemia (AML) 1st line patients are centered on the combination of Cytarabine and an anthracycline; Idarubicin (IDA), Daunorubicin (DNR), or Mitoxantrone (MIT). Patients may be treated with IDA, DNR, or MIT depending on the country of residence, because multiple clinical trials have not found significant differences among them. A new Personalized Medicine (PM) test developed by Vivia Biotech based on pharmacological responses in patient samples (ex vivo) is uncovering individual responses to these treatments. Our objective is to explore whether a significant % of individual patients may respond differently to IDA vs DNR vs MIT treatments, in spite that of their “on average” similar response shown by clinical trials. Patients and MethodsMulticenter, prospective, non-interventional study of the PETHEMA group for treatment of AML. Bone Marrow (BM) samples were collected at diagnosis for 160 AML patients. Samples were incubated for 48 hours in 96-well plates, each well containing different drugs or drug combinations, each at 8 different concentrations, enabling calculation of dose response curves for each single drug (CYT, IDA, DNR, MIT) and combination used in treatments (CYT-IDA, CYT-DNR, CYT-MIT). Drug response was evaluated as depletion of AML malignant cells in each well after 48 hours incubations. Annexin V-FITC was used to quantify the ability of the drugs to induce apoptosis. Malignant cells were identified with monoclonal antibodies and light scatter properties. 1) We use the whole bone marrow sample, retaining the erythrocyte population and serum proteins, during the entire incubation period; and after 48 h leukocytes are isolated prior to evaluation by flow cytometry. 2) We have pioneered development of a proprietary automated flow cytometry platform called ExviTech. 3) Pharmacological responses are calculated using pharmacokinetic population models. ResultsFigure left panel shows dose responses for both IDA (red) and DNR (blue) in 125 AML patient samples. Although their average curves (thick red & blue) are similar, the interpatient variability of either drug is quite large. We hypothesized that some patients could show very differential sensitivities to both drugs, as illustrated by the green arrow where a patient sample is resistant to DNR (right shifted dose response curve) but sensitive to IDA (left shifted dose response curve). To identify these cases Figure right panel shows a comparison of the potency IDA vs DNR. Potency is represented by their EC50 (concentration that kills 50% of the cells). Most dots tend to line up, but red dots represent patient samples with a difference in potency between these drugs >30%. Repeating this exercise for IDA-MIT and DNR-MIT to cover all alternatives among the 3 anthracyclines identifies 40% of patients samples with >30% different potency among IDA-DNR-MIT. Repeating this exercise with the combination treatments CYT-IDA, CYT-DNR, CYT-MIT increases to 58% the population of patients whose samples have a differential sensitivity to these anthracyclines. A fraction of this 57% of patients may benefit in if treatment selection among these 3 treatments were to be aided by this ex vivo testing sensitivities. To identify which fraction would benefit we would need a trial specifically designed. ConclusionsThis preliminary results show that Vivia's PM test seems able to identify a subset of AML patients who's ex vivo pharmacological response to anthracycline drugs is significantly different. Because this ex vivo test accurately predicts the clinical response to CYT-IDA, if these selective anthracycline ex vivo responses translate to clinical responses, a fraction of this 57% subpopulation could benefit significantly from receiving 1st or 2nd line treatments based on either IDA, DNR, MIT, and their combinations. Hence this approach stands for European integration of treatment protocols, based on ex vivo individual responses data rather than nationality. [Display omitted] Disclosures:Primo:Vivia Biotech: Employment. Hernandez-Campo:Vivia Biotech: Employment. Rojas:Vivia Biotech: Employment. Bennett:Vivia Biotech: Employment. Liebana:Vivia Biotech: Employment. Lopez:Vivia Biotech: Employment. Ballesteros:Vivia Biotech: Equity Ownership.

Leukemic Stem Cell Quantification Is Of Prognostic Value In Newly Diagnosed Patients In Chronic Phase Chronic Myeloid Leukemia (CML-CP) Receiving Nilotinib Therapy: Results From The ENEST1st Stem Cell Substudy

BackgroundLeukemic stem cells (LSCs) are considered to be very important therapeutic targets in CML, as it has been shown both in vitro and in vivo that tyrosine kinase inhibitor (TKI) therapy is not able to eradicate these entirely. We previously reported that LSC burden at diagnosis has significant prognostic impact on therapy outcome in imatinib and dasatinib treated CML patients. The ENEST1st study (NCT01061177) is focused on examining the role of first-line nilotinib therapy in patients in CML-CP. This translational, multinational, multicenter ENEST1st substudy addresses the predictive value of the stem cell profile in these patients. Patients and methodsBone marrow (BM) and/or peripheral blood (PB) samples were collected from newly diagnosed CML-CP patients before and after 1 and 3 months of nilotinib treatment. The LSC burden was analyzed by FISH and flow cytometry (FC). With the FISH method, CD34+ cells were pre-selected with paramagnetic beads after which they were fractionated into CD38+ (upper 80%, “progenitor”) and CD38- (lowest 5%, “stem cell”) pools using flow sorting. The proportion of Ph+ cells was assayed by counting 1,000 cells with interphase FISH using specific BCR-ABL1 probes. In the FC method, the stem cell compartment was defined as the lowest 1% of CD34+CD38- cells. LSCs were distinguished from normal hematopoietic stem cells (nHSCs) by either higher light scatter properties, and/or aberrant expression of CD7, CD11b and CD56, and/or higher CD45 and CD90 expression. Patient samples were assigned to as “residual nHSCs present” or “no residual nHSC present” at diagnosis and as “residual LSCs present” or “no residual LSCs present” at 1 and 3 months. Altogether, 48 patients from 6 European countries were investigated. ResultsBy FISH analysis at diagnosis, the proportion of BCR-ABL+ cells in the stem cell compartment varied markedly in individual patients (1%-100%) and was lower (85%) than in progenitor (96%) or whole BM fractions (96%, p<0.05). This LSC burden correlated significantly with high WBC count (r=0.44, p=0.008), hemoglobin (r=-0.51, p=0.002), spleen size (r=0.51, p=0.002) and PB (r=0.57, p=0.0002) and BM (r=0.55, p=0.0005) blast percentage. Weak correlation was also found with Sokal score (r=0.37, p=0.03). By FC method, similar to FISH, the range of LSCs was 0%-100% with a median of 73%. Forty percent of patients had detectable residual nHSC at diagnosis. These patients had significantly lower Sokal score (p=0.001), smaller spleen size (p=0.006), lower platelet count (p=0.04) and lower PB blast percentage (p=0.001). With both methods no prognostic relationship existed between LSC burden and eosinophil/basophil counts. Interestingly, by FISH method, the LSC burden at diagnosis correlated also with BCR-ABL transcript levels according to the international scale at 3 (r=0.39, p=0.03), 9 (r=0.41, p=0.01) and 18 months (r=0.42, p=0.02). Borderline significant associations were also found at 6 (p=0.06) and 12 (p=0.07) months. All patients lacking major molecular remission at 12 or 18 months, had >80% of BCR-ABL+ cells in the stem cell fraction at diagnosis. When LSCs were analyzed by the FC method, patients with residual nHSCs at diagnosis had significantly lower BCR-ABL levels at 3 (0.27 vs 1.74, p=0.025) and 6 (0.13 vs 1.21, p=0.03) months, but this was not reflected in lower BCR-ABL levels at 12 or 18 months. During nilotinib therapy, the proportion of BCR-ABL+ cells by FISH decreased rapidly both the in progenitor and stem cell compartments. At 3 months the median LSC percentage was 0.28%, and it did not differ significantly from the values detected either in the progenitor fraction (0.34%) or whole BM (0.29%). Similarly, only a few patients had detectable LSCs left during follow-up time-points with the FC method. ConclusionsLSC burden at diagnosis reflects the biology of the disease in newly diagnosed CML-CP patients. It also carries a prognostic value in first-line treated nilotinib patients, and a substantial number of patients not meeting the optimal response criteria at the 12-month time-point have high LSC burden at diagnosis. Furthermore, the presence of residual nHSCs at diagnosis is predictive for molecular response at 3 and 6 months. Nilotinib therapy markedly decreases LSC pool during the first 3 months of treatment. Noteworthy, the reduction is as potent in the LSC compartment as it is in more mature progenitor cell or whole BM fractions. Disclosures:Richter:Novartis: Consultancy, Research Funding, Speakers Bureau; Bristol Myers Squibb: Consultancy, Speakers Bureau. Fioretos:Novartis: unrestricted research grant Other. Giles:Novartis: Consultancy, Research Funding. Hochhaus:Novartis: Consultancy, Honoraria, Research Funding, Travel Other; BMS: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Ariad: Consultancy, Honoraria. Ossenkoppele:Novartis: Consultancy, Research Funding, Speakers Bureau; Bristol Myers Squibb: Consultancy, Speakers Bureau. Porkka:Novartis: Consultancy, Research Funding, Speakers Bureau; BMS: Consultancy, Research Funding, Speakers Bureau. Wolf:Novartis: Honoraria, Research Funding; Pfizer: Honoraria; Bristol-Meyers Squibb: Honoraria. Janssen:Novartis: Consultancy, Research Funding. Mustjoki:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau.

Multiparameter flow cytometry is necessary for detection, characterization and diagnostics of composite mature B-cell lymphoproliferative neoplasms

Composite mature B-cell lymphoproliferative neoplasms are rare entities characterized by the simultaneous presence of two or more distinctive B-cell derived monoclonal malignancies. This retrospective study used multiparametric flow cytometric analysis aimed at immunophenotypic profiling of composite mature B-cell lymphoproliferative neoplasms in a cohort of 413 subsequent patients with de novo leukemic B-cell chronic lymphoproliferative disorders diagnosed in our institution during a 30-month period. Biclonality was found in 16 (3.9 %) patients. The vast majority (88 %) of the cases had one of the clones phenotypically corresponding to chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Only when composite cases were categorized by phenotype of the non-CLL/SLL malignant population did we find a statistically significant (P = 0.001) higher frequency of biclonality among cases with hairy cell leukemia (22 %). Biclonal cases had the overall B-cell membrane κ to λ ratio within the normal range (median, 1.9; reference interval 0.5-4.0), making recognition of malignancy somewhat challenging. Our analysis strategy was therefore based on the detection of aberrant B-cell phenotypes, with subsequent confirmation of the monoclonal nature of neoplastic clones with regards to light chain restriction analysis. Discrimination of the coexisting clones in biclonal cases was possible on the basis of the expression of other antigen(s) (63 %), light scatter properties (44 %), different surface light chain restriction (69 %) and/or pattern of expression (44 %). The most informative cell surface antigens proved to be CD22, CD20, surface IgM, and CD23. In conclusion, historic κ/λ ratio is not a reliable approach and is a poor measurement for the detection of composite lymphomas. More creative analysis techniques should be utilized for this purpose.

Enumeration of platelets by multiparameter flow cytometry using platelet-specific antibodies and fluorescent reference particles.

The correct enumeration of platelets is still an elusive matter. This is mainly due to the fact that commercial instruments which are used for platelet counting cannot discriminate platelets from other cellular particles and precipitates that cause similar signals. Visual (chamber counting) methods are still frequently used in routine laboratories to verify low automated platelet counts (< 50 x 10/l) despite obvious technical and statistical drawbacks. The following report shows how platelet counts can be measured by multiparameter flow cytometry with the help of reference particles (fluorescent latex beads) and platelet-specific antibodies i.e. anti-GPIIb/IIIa(CD41a), anti-GP Ib-alpha (CD42b) and anti-GP IIIa (CD61). The linearity of this method was highly satisfactory and the observed imprecision was within acceptable limits. At a platelet concentration of 10 x 10(9)/l the coefficient of variation (CV, n = 10) ranged from 5.3% (PCV = 0.456) to 5.6% (PCV = 0.148). Accuracy was evaluated by comparing results to the ICSH-selected method for platelet counting. The correlation of both methods was significant (P < 0.005) and Passing-Bablok's linear regression analysis showed no systematic differences between the two methods. Comparisons of this new platelet counting technique were also performed with routine visual methods, automated blood analysers (Technicon H-1, Sysmex E-5000) and a different flow cytometric method using only forward and side light scatter properties of platelets for their discrimination. The linear correlation of all methods was significant (P < 0.01) at platelet concentrations above 50 x 10(9)/l. At lower platelet concentrations, our new platelet counting technique correlated significantly only with the visual and the forward/side scatter methods.(ABSTRACT TRUNCATED AT 250 WORDS)

Statistical classification of multivariate flow cytometry data analyzed by manual gating: Stem, progenitor, and epithelial marker expression in nonsmall cell lung cancer and normal lung

The use of supervised classification to extract markers from primary flow cytometry data is an emerging field that has made significant progress, spurred by the growing complexity of multidimensional flow cytometry. Whether the markers are extracted without supervision or by conventional gate and region methods, the number of candidate variables identified is typically larger than the number of specimens (p > n) and many variables are highly intercorrelated. Thus, comparison across groups or treatments to determine which markers are significant is challenging. Here, we utilized a data set in which 86 variables were created by conventional manual analysis of individual listmode data files, and compared the application of five multivariate classification methods to discern subtle differences between the stem/progenitor content of 35 nonsmall cell lung cancer and adjacent normal lung specimens. The methods compared include elastic-net, lasso, random forest, diagonal linear discriminant analysis, and best single variable (best-1). We described a broadly applicable methodology consisting of: 1) variable transformation and standardization; 2) visualization and assessment of correlation between variables; 3) selection of significant variables and modeling; and 4) characterization of the quality and stability of the model. The analysis yielded both validating results (tumors are aneuploid and have higher light scatter properties than normal lung), as well as leads that require followup: Cytokeratin+ CD133+ progenitors are present in normal lung but reduced in lung cancer; diploid (or pseudo-diploid) CD117+CD44+ cells are more prevalent in tumor. We anticipate that the methods described here will be broadly applicable to a variety of multidimensional cytometry problems.

Role of Paroxysmal Nocturnal Hemoglobinuria (PNH) Clones in Refractory Anemias

AbstractAbstract 4406 Background:The presence of paroxysmal nocturnal hemoglobinuria (PNH) clones in the setting of aplastic anemia (AA) and myelodysplastic syndrome (MDS) highlights the pathogenetic link between these disorders. The awareness that small clones of PNH cells could be detected by flow cytometry in aplastic anaemia patients who had negative Ham test has led to classifying PNH into two broad groups such as Haemolytic PNH which can have 50% presenting with thrombosis and a hypo plastic group which behaves like aplastic anaemia. Even if there is a small PNH clone in the setting of aplastic anaemia, then the patients respond better to immunosuppressive therapy.Flowcytometry is a relatively new advanced technique in many parts of India and there is no widespread consensus among methodology. Further, the clinical and prognostic significance of monitoring PNH clone size by serial flow cytometry is yet to be established here. Objective:To validate the role of flowcytometry in the diagnosis of PNH, in the setting of aplastic anaemia, myelodysplastic syndrome or abdominal or cerebral vein thrombosis in the setting of PNH which may help to prognosticate the course and treatment. Material and methods:The study was both retrospective and prospective. The prospective arm of the study was conducted from September 2009 to April 2010. Ham's test and Sucrose lysis test were done as screening tests and were reported as positive or negative. Other laboratory studies including hemogram and bone marrow analysis were done as part of standard care. In the retrospective group there were 32 patients who were diagnosed as PNH based on Ham's and Sucrose lysis test followed up from 1990.Four color flowcytometry immunophenotypic analysis with CD55 and CD59 was used to detect PNH clones in RBCs. EDTA-anticoagulated whole blood was stained with anti–CD 55-Phycoerythrin (PE) and anti–CD 59-PE.A minimum of 10,000 events were acquired. For analysis, RBCs were identified by light-scatter properties. For the purpose of the study, 3% of type II and/or type III cells were considered as threshold to diagnose PNH clones in both CD55 and CD 59 gated population. The results of laboratory studies including transfusion history and flow cytometry were correlated. Institutional ethical committee approval was obtained for the conduct of the study. Results:33 patients were included as part of the prospective group. The number of males was more compared to females (19:14). 78% of patients had hemoglobin of less than 7gm%. The presentation varied from unexplained anemia to thrombosis. Based on flow cytometry, 3 patients were found to have PNH clones in the RBC lineage by both CD 55 and CD 59. CD 55 alone picked up one more patient. In the retrospective group there were 32 patients who were followed up from 1990. Interestingly, two of them who had earlier presented with aplastic anemia had transformed into PNH in a span of 3years and 6 months. All patients were treated with Stanazolol and cyclosporine. The median survival was 12.14. months with Kaplan-Meier survival estimates of 30 and 20 percent at 121 months, and 143 months after diagnosis, respectively. The patients in the prospective group are being followed up. Discussion:The Ham test, sucrose lysis test, and modified Ham tests rely on the differential sensitivity of PNH red cells to haemolysis. These tests, although suitable for haemolytic PNH, cannot reliably detect small populations of PNH red cells nor differentiate partially and completely deficient cells. One patient who presented with DVT in whom increase in clone size was demonstrated by serial flowcytometry. Testing for the presence of PNH RBC clones provides additional diagnostic information and the thrombotic event may probably directly related to the size of the PNH clone.Conclusions: Our data demonstrate the utility of RBC assay in flow cytometry in the primary screening of PNH, AA, and MDS patients. The clinical and prognostic significance of monitoring PNH clone size by serial flow cytometry is relatively new and could be demonstrated in one patient. Disclosures:No relevant conflicts of interest to declare.

Flow-Cytometric Isolation and Enrichment of Teleost Type A Spermatogonia Based on Light-Scattering Properties1

The transplantation of germ cells is a powerful tool both for studying their development and for reproductive biotechnology. An intraperitoneal germ cell transplantation system was recently developed for use in several teleost species. Donor germ cells transplanted into the peritoneal cavity of hatchlings migrated toward and were incorporated into the recipient's genital ridges, where they underwent gametogenesis. Among male germ cells, only type A spermatogonia were capable of colonizing the recipient gonads, unlike those at more advanced stages. The enrichment of type A spermatogonia is therefore important to achieve efficient donor-cell incorporation and subsequent donor-derived gametogenesis. Here we established a simple and rapid system of isolation and enrichment for fish type A spermatogonia, using flow cytometry. Type A spermatogonia were found to have distinctive forward and side light scatter properties compared to that with other types of testicular cell. Based on these characteristics, we were able to isolate and enrich type A spermatogonia by using flow cytometry. After intraperitoneal transplantation, the enriched type A spermatogonia could be successfully incorporated into the recipient genital ridges. This flow cytometry approach using forward and side light scatter was also found to be applicable to other salmonid and sciaenid species, suggesting that it could be a powerful tool for isolating and enriching transplantable type A spermatogonia in a wide range of teleosts. We expect this method to contribute significantly to germ cell biology and biotechnology.

Case study interpretation-Portland: Case 2

Case study interpretation-Portland: Case 2

Altered immunophenotypic features of peripheral blood platelets in myelodysplastic syndromes

Multiparameter flow cytometric analysis of bone marrow and peripheral blood cells has proven to be of help in the diagnostic workup of myelodysplastic syndromes. However, the usefulness of flow cytometry for the detection of megakaryocytic and platelet dysplasia has not yet been investigated. The aim of this pilot study was to evaluate by flow cytometry the diagnostic and prognostic value of platelet dysplasia in myelodysplastic syndromes. We investigated the pattern of expression of distinct surface glycoproteins on peripheral blood platelets from a series of 44 myelodysplastic syndrome patients, 20 healthy subjects and 19 patients with platelet alterations associated to disease conditions other than myelodysplastic syndromes. Quantitative expression of CD31, CD34, CD36, CD41a, CD41b, CD42a, CD42b and CD61 glycoproteins together with the PAC-1, CD62-P, fibrinogen and CD63 platelet activation-associated markers and platelet light scatter properties were systematically evaluated. Overall, flow cytometry identified multiple immunophenotypic abnormalities on platelets of myelodysplastic syndrome patients, including altered light scatter characteristics, over-and under expression of specific platelet glycoproteins and asynchronous expression of CD34; decreased expression of CD36 (n = 5), CD42a (n = 1) and CD61 (n = 2), together with reactivity for CD34 (n = 1) were only observed among myelodysplastic syndrome cases, while other alterations were also found in other platelet disorders. Based on the overall platelet alterations detected for each patient, an immunophenotypic score was built which identified a subgroup of myelodysplastic syndrome patients with a high rate of moderate to severe alterations (score>1.5; n = 16) who more frequently showed thrombocytopenia, megakaryocytic dysplasia and high-risk disease, together with a shorter overall survival. Our results show the presence of altered phenotypes by flow cytometry on platelets from around half of the myelodysplastic syndrome patients studied. If confirmed in larger series of patients, these findings may help refine the diagnostic and prognostic assessment of this group of disorders.