Side Scatter Plot Research Articles

Lecithin - Bifidobacterium probiotics interactions: a case study

Probiotic formulations containing lecithin phospholipids have been shown to extend their viability. However, the protective effect of lecithin and the properties of the mixtures of lecithin and probiotic cells in aqueous media have not been fully documented. The aim of this study was to investigate the interactions between Bifidobacterium animalis ssp. lactis (Bifido) probiotics and lecithin. The lecithin-Bifido interactions were dependent on lecithin concentration. FTIR and Raman spectroscopy suggested that Bifido cells interacted with lecithin molecules, mainly through hydrophobic interactions. At the lecithin concentration of 0.1%.w/v, Bifido lost only about 17% of their viability over 56 days, while free Bifido probiotics (without lecithin) lost about 66% of their viability in the same time period. Furthermore, flow cytometer side scatter plots also showed that Bifido-lecithin samples at 0.1.%w/v lecithin, showed the lowest side scatter, which is indicative of lower cellular stress and a more stable environment to preserve Bifido cells viability over time.

Monocyte platelet aggregates in children with Kawasaki disease- a preliminary study from a tertiary care centre in North-West India

BackgroundPlatelet activation is an integral part of pathogenesis of Kawasaki disease (KD). However, there is paucity of literature on flow-cytometry based assessment of platelet activation in KD. We aimed to analyse monocyte-platelet aggregates (MPAs), one of the sensitive markers for platelet activation, by flow cytometry in children with KD.FindingsIn this single-centre prospective study, we have enrolled 14 children with KD and results were compared with age-matched febrile (n = 15) and healthy (n = 13) controls. After gating monocytes in side-scatter plot, MPAs were identified based on CD14 and CD41 expression. Two (2) ml of blood samples for children with KD were collected at 3 phases of illness- acute stage before start of intravenous immunoglobulin or aspirin, 24 h after completion of IVIg infusion, and 3 months after acute episode of KD.Children with KD had a significantly higher MPA% values [Median (IQR)- 41.3% (26.6, 52.7)] when compared with febrile [Median (IQR)- 5.98% (2.98-9.72)] and normal [Median (IQR)- 4.48% (2.57-5.59)] controls, p<0.01. On follow-up, the MPA% showed a gradual decline in children with KD, but even at 3 months, the value [Median (IQR)- 7.55% (4.15-14.6)] was higher compared to healthy controls [Median (IQR)- 4.48% (2.57-5.59)].ConclusionsOur results suggest that MPA% was significantly elevated in acute stages in children with KD and activated platelets may continue to persist even after systemic inflammation has subsided. Future studies are warranted whether objective evidence of platelet activation may guide the use of immunomodulatory and anti-platelet therapy in KD.

Issue Highlights - November 2020.

Issue Highlights - November 2020.

Plasmacytoid Dendritic Cell Levels at End of Induction Are Predictive of MRD Clearance and Also Influence Disease Outcome in a Subset of Patients

Introduction:Plasmacytoid dendritic cells (pDCs) are a subset of immune cells that secrete type 1 interferons and serve as antigen presenting cells. In many tumors increased pDC frequencies have been observed and are involved in tumor response initiation. However, there is not much data in acute myeloid leukemia especially in the context of minimal residual disease and outcome. Here we evaluated the frequencies of pDCs in the post induction bone marrow and found a significant correlation with post induction MRD levels. Furthermore, we found that high levels of pDCs in MRD positive patients confer a better overall survival (OS).Methods:All adult (>18 years ) of patients who were treated for AML [other than AML with t(15;17)] were accrued over a 2 year period. All patients received "3+7" induction therapy with daunorubicin and cytarabine. If patients were in morphological CR at end of induction, they either received 3 courses of 12-18 gm/m2 high dose cytarabine (HiDAC) or aBMT if feasible. The presence of MRD was assessed using 8 colour flow cytometry on a post induction bone marrows using CD45, CD36, CD38, CD123, CD33, CD117, CD34, HLADR, CD7, CD56, CD13, CD19, CD16, CD11b, CD15 and CD14. Minimum of 500,000 events were acquired/tube on an 8 colour BD FACS Canto II or a 10 colour BC Navios instruments. Kaluza software (v1.3) was used to analyze the .fcs files. MRD was calculated as a percentage of abnormal leukemic cells per total viable cells as gated in forward scatter v side scatter plot. pDCs were calculated as CD123 bright population which expressed HLA-DR (while gating on the progenitors and monocytes based on their expression of CD45 and side scatter). The pDC percentages were counted as a fraction of all viable cells. pDCs were divided into pDC High and pDC Low groups, based on a median pDC value of 0.1%. OS was calculated from start of induction therapy to time of last follow up or death. Chi Squared test was used to analyse categorical values of pDC (hi v/s low) and MRD (positive v/s negative). Log rank test was used to compare pDC groups in MRD positive subset.Results:At a median follow up of 16 months, OS was 47.8%. After exclusion of induction deaths, a total of 94 patients of adult AML was assessed for the presence of MRD at the end of induction. Of these MRD was detected in 48 (51.1%, range 0.01-40%). pDC values ranged from <0.01% to 1.95% (median 0.15%). Median pDC value in the MRD positive group was lower (0.05%) as compared to the MRD negative group (0.23%). Out of 94 patients of 44.7% patients belonged to the pDC Low group of which majority patients were MRD positive (66.7% of the pDC Low group). Similarly, majority of patients in the pDC High group were MRD negative (61.5% of the pDC High group). A statistical analysis of these categories was found to be significant (p=0.008). When pDCs (high/low) groups were analyzed in each of MRD subsets we found a significant association in MRD positive group (p=0.03), indicating that MRD positive patients who have low pDC levels have a worse OS (median OS: 10.3 months).Conclusion:These pilot data seem to indicate that the pDC burden in the bone marrow may have a role in influencing MRD clearance in the bone marrow. A detailed investigation of the pDC function in the bone marrow microenvironment is warranted. Functional studies on pDCs are warranted to understand the dynamics of disease relapse. [Display omitted] DisclosuresPatkar:Wellcome Trust-DBT India Alliance: Research Funding.

Plasmacytoid Dendritic Cell Burden in the Bone Marrow Predicts End of Induction Minimal Residual Disease Status in Adult Acute Myeloid Leukemia

Introduction:Plasmacytoid dendritic cells (pDCs) are a subset of immune cells that secrete type 1 interferons and serve as antigen presenting cells. In many tumors increased pDC frequencies have been observed and are involved in tumor response initiation. However, there is not much data in acute myeloid leukemia especially in the context of minimal residual disease. Here we evaluated the frequencies of pDCs in the post induction bone marrow and found a significant correlation with MRD levels.Methods:All adult (>18 years ) of patients who were treated for AML [other than AML with t(15;17)] were accrued over a 2 year period. The presence of MRD was assessed using 8 colour flow cytometry on a post induction bone marrows using CD45, CD36, CD38, CD123, CD33, CD117, CD34, HLADR, CD7, CD56, CD13, CD19, CD16, CD11b, CD15 and CD14. Minimum of 500,000 events were acquired/tube on an 8 colour BD FACS Canto II or a 10 colour BC Navios instruments. Kaluza software (v1.3) was used to analyze the .fcs files. MRD was calculated as a percentage of abnormal leukemic cells per total viable cells as gated in forward scatter v side scatter plot. pDCs were calculated as CD123 bright population which expressed HLA-DR (while gating on the progenitors and monocytes based on their expression of CD45 and side scatter). The pDC percentages were counted as a fraction of all viable cells. Based on the results the levels of pDCs were divided into pDC High and pDC Low groups. Statistical analysis was done using Chi squared groups.Results:After exclusion of induction deaths, a total of 94 patients of adult AML was assessed for the presence of MRD at the end of induction. Of these MRD was detected in 48 (51.1%, range 0.01-40%). pDC values ranged from <0.01% to 1.95% (median 0.15%). Median pDC value in the MRD positive group was lower (0.05%) as compared to the MRD negative group (0.23%). Out of 94 patients of 44.7% patients belonged to the pDC Low group of which majority patients were MRD positive (66.7% of the pDC Low group). Similarly majority of patients in the pDC High group were MRD negative (61.5% of the pDC High group). A statistical analysis of these categories was also found to be significant (p=0.008)Conclusion:These pilot data seem to indicate that the pDC burden in the bone marrow may have a role in influencing MRD clearance in the bone marrow. A detailed investigation of the pDC function in the bone marrow microenvironment is warranted. DisclosuresNo relevant conflicts of interest to declare.

Dual kappa and lambda expressing in mature B-cell neoplasm: An unusual case

A 57 years old male presented with transfusion dependent anemia for last 2 months with mild weight loss and on and off fever. On examination he was found to have small axillary lymph nodes with moderate splenomegaly. Flow cytometry revealed 63.2% cells in the lymphocyte window in cluster of differentiation (CD) 45 versus side scatter plot. Further analysis on CD-19 gated cells showed that - 75.9% of these cells expressed CD-19 plus the expression of CD23, CD25, CD43, CD20 and CD22. The entire cluster showed dual expression of kappa and lambda light chains. Final diagnosis of low-grade B non-Hodgkin lymphoma was made. Given that dual kappa and lambda expressing in mature B cell neoplasm has been described in literature, it is important to differentiate such cases from reactive lymphoid proliferations.

Abstract 5512: Zirconium phosphate nanoparticle matrix as potential delivery system for cisplatin to cancer cells in vitro

Abstract Cisplatin (cis-diamminedichloroplatinum(II), have been shown to be active against ovarian and testicular cancer, Hodgkin's lymphoma, and certain other malignancies, however, its clinical effectiveness is limited by significant side effects and the emergence of drug resistance. Delivery of anticancer drugs such as cisplatin to the tumor cells without damaging healthy organs or tissues is highly difficult if not impossible. In the last several years nanoparticle-mediated drug delivery is being studied as a novel valuable mean for overcoming this problem. We developed a novel nanoparticle intercalated formulation of cisplatin on a layered matrix of zirconium phosphate (ZrP). It was expected that cisplatin, while being excluded from the external medium within ZrP layers will remain biologically inactive and that cisplatin will be released selectively in the tumor cell. The purpose of this study was to determine the cytotoxicity and effectiveness of ZrP nanoparticles in the delivery of cisplatin. Cell viability was measured by MTT assay. Flow cytometry analysis was used to determine cisplatin-induced apoptosis, and cell cycle distribution. ZrP alone did not affect the viability of breast cancer cells (MCF-7), human leukemic lymphoblast (CEM) and human peripheral blood lymphocytes (PBLs) after 24 hours in contact with different concentrations of ZrP. Cisplatin intercalated into ZrP at the highest concentration (30 μM cisplatin) reduced in 40% the cell viability of MCF-7 and CEM. The IC50 of cisplatin alone in MCF-7 was 10 μM. The side scatter plot showed that normal PBLs scatter plot is different from PBLs that have been in contact with cisplatin-intercalated ZrP for 18 hours. These PBLs carrying ZrP were displaced to the right quadrant suggesting an increased granularity that can be associated to the incorporation of ZrP nanoparticles. Moderate cisplatin-induced apoptosis was evident in cells treated with ZrP-intercalated cisplatin for 18 hours. The cell cycle analysis showed that ZrP alone does not cause cell cycle arrest. Our results suggest that application of ZrP nanoparticle-mediated drug transport in cancer chemotherapy is highly promising and more studies are warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5512.

Arginase Activity Mediates Retinal Inflammation in Endotoxin-Induced Uveitis

Arginase has been reported to reduce nitric oxide bioavailability in cardiovascular disease. However, its specific role in retinopathy has not been studied. In this study, we assessed the role of arginase in a mouse model of endotoxin-induced uveitis induced by lipopolysaccharide (LPS) treatment. Measurement of arginase expression and activity in the retina revealed a significant increase in arginase activity that was associated with increases in both mRNA and protein levels of arginase (Arg)1 but not Arg2. Immunofluorescence and flow cytometry confirmed this increase in Arg1, which was localized to glia and microglia. Arg1 expression and activity were also increased in cultured Muller cells and microglia treated with LPS. To test whether arginase has a role in the development of retinal inflammation, experiments were performed in mice deficient in one copy of the Arg1 gene and both copies of the Arg2 gene or in mice treated with a selective arginase inhibitor. These studies showed that LPS-induced increases in inflammatory protein production, leukostasis, retinal damage, signs of anterior uveitis, and uncoupling of nitric oxide synthase were blocked by either knockdown or inhibition of arginase. Furthermore, the LPS-induced increase in Arg1 expression was abrogated by blocking NADPH oxidase. In conclusion, these studies suggest that LPS-induced retinal inflammation in endotoxin-induced uveitis is mediated by NADPH oxidase-dependent increases in arginase activity.

The usefulness of CD64, other monocyte-associated antigens, and CD45 gating in the subclassification of acute myeloid leukemias with monocytic differentiation.

Immunophenotyping by flow cytometry is widely used in the diagnosis and subclassification of acute myeloid leukemia (AML). CD14 is the monocyte-associated antigen most widely used to identify AML with monocytic differentiation (French-American-British classes M4 and M5); however, we observed that CD14 expression is frequently diminished or absent in such cases. To identify monocyte-associated antigens that might improve recognition of AML M4 and M5, we used 3-color flow cytometry and a panel of antibodies reported to distinguish cells of monocytic lineage in 44 cases of AML. In addition, CD45 vs logarithmic side scatter plots were analyzed in all cases. As expected, CD14 was highly specific but was only moderately sensitive for monocytic differentiation. CD64 had the best-combined sensitivity and specificity for AML M4 and M5. CD45 vs logarithmic side scatter analysis showed a higher percentage of monocytes in AML M4 and M5 compared with nonmonocytic AML. CD64 was expressed in 5 of 5 cases of acute promyelocytic leukemia (AML M3), but the intensity of staining was significantly less in AML M3 than in AML M4 and M5. Our findings show that addition of CD64 and CD45 vs logarithmic side scatter analysis to CD14 greatly improves flow cytometric detection of AML with monocytic differentiation and that CD64, also expressed in AML M3, may help distinguish AML M3 from other subtypes.

Identification of eosinophils in lysed whole blood using side scatter and CD16 negativity

The identification of eosinophils in lysed whole blood by flow cytometry can be problematic, since these cells overlap significantly with the neutrophil cluster on forward scatter versus side scatter plots of whole blood samples. Current methods can be time-consuming when running multiple samples or may compromise yield in the interests of greater accuracy. The use of eosinophil purification techniques prior to FACS analysis or sorting as a way of ensuring purity may have unpredictable effects on eosinophil activation, leading to questionable data interpretation. Here we describe a simple, single-step method for definition of eosinophils utilizing their high side scatter and CD16 fluorescence negativity to differentiate them from neutrophils. The purity of the neutrophil and eosinophil populations sorted with this gate is close to 100% regardless of the peripheral blood eosinophil count, while the population obtained by sorting on a plot of FSC/SSC was a mixture of eosinophils and neutrophils. We suggest this method as a simple, reproducible, and accurate way of defining eosinophils by flow cytometry for analysis or sorting.

Identification of eosinophils in lysed whole blood using side scatter and CD16 negativity

The identification of eosinophils in lysed whole blood by flow cytometry can be problematic, since these cells overlap significantly with the neutrophil cluster on forward scatter versus side scatter plots of whole blood samples. Current methods can be time-consuming when running multiple samples or may compromise yield in the interests of greater accuracy. The use of eosinophil purification techniques prior to FACS analysis or sorting as a way of ensuring purity may have unpredictable effects on eosinophil activation, leading to questionable data interpretation. Here we describe a simple, single-step method for definition of eosinophils utilizing their high side scatter and CD16 fluorescence negativity to differentiate them from neutrophils. The purity of the neutrophil and eosinophil populations sorted with this gate is close to 100% regardless of the peripheral blood eosinophil count, while the population obtained by sorting on a plot of FSC/SSC was a mixture of eosinophils and neutrophils. We suggest this method as a simple, reproducible, and accurate way of defining eosinophils by flow cytometry for analysis or sorting. Cytometry 30:313–316, 1997. © 1997 Wiley-Liss, Inc.

A new flow cytometric method for discrimination of apoptotic cells and detection of their cell cycle specificity through staining of F‐actin and DNA

Drug-initiated apoptosis of human leukemia HL-60, THP-1, and U-937 cells was studied via multiparameter flow cytometry and cell sorting. A new flow cytometric method that allows both identification and quantitation of apoptotic cells and estimation of their cell cycle specificity is presented. The method is based on paraformaldehyde fixation followed by staining of F-actin and DNA with fluorescein isothiocyanate (FITC)-phalloidin and propidium iodide (PI), respectively. Bivariate green fluorescence (F-actin) vs. side scatterplots of HL-60 cells treated with 10 microM etoposide for 4 h showed two cell populations, one with high green fluorescence and low side scatter and one with low green fluorescence and high side scatter. Sorting revealed cells with intact nuclei in the high green fluorescence/low side scatter population and cells with fragmented nuclei in the low green fluorescence/high side scatter population, demonstrating that the cells in the latter population were apoptotic. Exposure of HL-60 cells to 10 microM etoposide for 4 h resulted in S-phase selective apoptosis, whereas 5 micrograms/ml cycloheximide initiated apoptosis mainly in G0/G1-phase and S-phase cells. The apoptotic response of HL-60 cells to 20 GY gamma-irradiation was selective for S-phase and G2 + M-phase cells. The present method offers the opportunity to estimate the cell cycle distributions of both the apoptotic and the nonapoptotic cell populations, which is especially valuable when apoptosis occurs in association with cell cycle perturbations. A similar shift from one to two cell populations in green fluorescence vs. side scatter-plots, similar to that observed for HL-60 cells, was observed in the THP-1 and U-937 cell lines secondary to etoposide treatment.