Cytometric Signatures Research Articles

Developing a Flow Cytometric Signature of Carfilzomib Responsiveness in Myeloma

Introduction: Multiple myeloma remains an incurable disease with a significant variation in therapeutic response. Choice of treatment is largely determined by prior lines of therapy and the patient's fitness. However, myeloma is a highly heterogeneous disease and this approach risks failing to deliver the right drug at the right time to an individual patient. To begin to address this, we have adopted machine learning approaches to predict individual responses to specific anti-myeloma therapies. We have previously shown that it is possible to select rationally between bortezomib- and lenalidomide-based therapy using a gene expression signature. Gene expression models are difficult to implement clinically, so more recently we presented a five gene mutational signature to identify carfilzomib-specific responses. Although easier to translate, such molecular signatures can still be hindered clinically by slow processing and technical failures. Because flow cytometry is well established in the diagnostic laboratory and is sensitive enough to detect minimal residual disease, we set out to develop an immunophenotypic signature of carfilzomib-specific responsiveness in primary myeloma. Methods We originally developed our mutational signature on whole exome sequencing of diagnostic samples from the CARDAMON clinical trial. For the current study, we performed RNA sequencing on 80 of these. Patients were divided into carfilzomib-responsive and non-responsive groups according to their mutational signature. Genes were filtered for those encoding cell surface proteins. A second round of filtering selected only those genes whose RNA expression correlated well with protein expression as determined by plasma membrane fractionation and mass spectrometry. Genes differentially expressed between the two groups were then detected using DeSEQ2. Logistic regression on these candidate genes was performed to assess whether they could be used to correctly classify patients for carfilzomib responsiveness and hence form a predictive flow cytometric panel. Results 76 patients from the CARDAMON trial had RNAseq that passed quality control. 128 genes encoded cell surface proteins, were reliable predictors of cell surface protein expression (R2 > 0.8), and were differentially expressed between carfilzomib-responsive and non-responsive patients. No single cell surface protein alone could correctly classify patients. However, using multiple logistic regression, six genes whose expression could be detected by flow cytometry (CLEC7A, STRA6, TMPRSS11E, CD27, GPR176, TK1, LPXN) together predicted carfilzomib responsiveness with an AUC of 0.77. Independent validation of these genes by RNA-Seq in the CoMMpass dataset showed that they could predict carfilzomib responsiveness with an AUC of 0.803 (Figure 1A). Indeed, survival for those patients who were predicted to be carfilzomib-responsive by our potential flow panel was significantly higher than those predicted to be resistant (median 34 months vs 15months ; p= 0.0032), figure 1B. Conclusions By combining transcriptomic and proteomic approaches, we have generated a panel of six cell surface proteins that are amenable to flow cytometry and can predict carfilzomib-responsiveness. Prospective validation of these markers is underway. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A simple strategy for sample annotation error detection in cytometry datasets.

Mislabeling samples or data with the wrong participant information can affect study integrity and lead investigators to draw inaccurate conclusions. Quality control to prevent these types of errors is commonly embedded into the analysis of genomic datasets, but a similar identification strategy is not standard for cytometric data. Here, we present a method for detecting sample identification errors in cytometric data using expression of human leukocyte antigen (HLA) class I alleles. We measured HLA-A*02 and HLA-B*07 expression in three longitudinal samples from 41 participants using a 33-marker CyTOF panel designed to identify major immune cell types. 3/123 samples (2.4%) showed HLA allele expression that did not match their longitudinal pairs. Furthermore, these same three samples' cytometric signature did not match qPCR HLA class I allele data, suggesting that they were accurately identified as mismatches. We conclude that this technique is useful for detecting sample-labeling errors in cytometric analyses of longitudinal data. This technique could also be used in conjunction with another method, like GWAS or PCR, to detect errors in cross-sectional data. We suggest widespread adoption of this or similar techniques will improve the quality of clinical studies that utilize cytometry.

Community analysis of high‐ and low‐nucleic acid‐containing bacteria in NW Mediterranean coastal waters using 16S rDNA pyrosequencing

The ecological significance of the marine bacterial populations distinguishable by flow cytometry on the basis of the fluorescence (FL) of their nucleic acid (NA) content and proxies of cell size (such as side scatter, SSC) remains largely unknown. Some studies have suggested that cells with high NA (HNA) content and high SSC (HS) represent the active members of the community, while the low NA (LNA) cells are inactive members of the same phylogenetic groups. But group-specific activity measurements and phylogenetic assignment after cell sorting have suggested this is not be the case, particularly in open-ocean communities. To test the extent to which the different NA subgroups are similar, and consequently the extent to which they likely have similar ecological and biogeochemical roles in the environment, we analysed the phylogenetic composition of three populations after cell sorting [high NA-high SC (HNA-HS), high NA-low SC (HNA-LS), low NA (LNA)] by 454 pyrosequencing in two contrasting periods of the year in NW Mediterranean coastal waters (BBMO, Blanes Bay Microbial Observatory) where these three populations have recurrent seasonal patterns. Statistical analyses showed that summer and winter samples were significantly different and, importantly, the sorted populations within a sample were composed of different taxa. The majority of taxa were associated with one NA fraction only, and the degree of overlap (i.e. OTUs present simultaneously in 2 fractions) between HNA and LNA and between summer and winter communities was very small. Rhodobacterales, SAR116 and Bacteroidetes contributed primarily to the HNA fraction, whereas other groups such as SAR11 and SAR86 contributed largely to the LNA fractions. Gammaproteobacteria other than SAR86 showed less preference for one particular NA fraction. An increase in diversity was observed from the LNA to the HNA-HS fraction for both sample dates. Our results suggest that, in Blanes Bay, flow cytometric signatures of natural communities track their phylogenetic composition.

Anti-TNF-α therapy monitoring in chronic rheumatic diseases by cytometric profiling of peripheral blood cells

Ankylosing spondylitis (AS) is a chronic inflammatory disorder mainly affecting the axial skeleton and belongs to a group of rheumatic diseases known as spondyloarthritides. Anti-tumour necrosis factor (TNF)-α (tumour necrosis...

Eutrophication in a tropical pond: Understanding the bacterioplankton and phytoplankton dynamics during a vibriosis outbreak using flow cytometric analyses

In tropical shrimp ponds, the increasing of feed input, concomitantly with the stocking shrimp biomass, induces an eutrophication of the ecosystem. Although difficult to maintain, its stability is required to guarantee the success of the culture. A 110-day period of phytoplankton and bacterioplankton stock and dynamics in an earthen pond (1.2 ha area, 1 m depth) was monitored using flow cytometry to provide baseline information on community characteristics and ecosystem instability. Seven autotrophic cell types were identified over the whole sampling period. Prokaryotic cells included Synechococcus sp., a group named UNK which presented an atypical new flow cytometric signature and picoeukaryotes (PEUK). Nanophytoplankton cells were represented by 4 groups: NAN1, NAN2, NAN3 and Cryptophytes. During the first part of the survey, picophytoplankton dominated the phytoplanktonic assemblage. The mean abundance of total cells (up to 8 × 10 6 cells mL −1) was among the highest recorded in marine and brackish waters. Bacterial abundance and production ranged from 0.8 to 5.1 × 10 7 cells mL −1 and from 30 to 110 μg C L −1 h −1. A shift from pico to nanophytoplankton abundance was observed for a few days from d 96. During this period, heterotrophic bacteria production and abundance suddenly dropped, implying a change in the functioning of the microbial loop. This shift was concomitant with a significant shrimp mortality outbreak due to Vibrio penaeicida, the etiological agent of a disease known as Syndrome 93, which affects the shrimp industry in New Caledonia. This survey suggests that flow cytometric analysis could be used for the monitoring of aquaculture systems to improve our understanding of the complex phytoplankton and bacterial dynamics of these systems and its potential influence on disease development.

Significant CO2 fixation by small prymnesiophytes in the subtropical and tropical northeast Atlantic Ocean

Global estimates indicate the oceans are responsible for approximately half of the carbon dioxide fixed on Earth. Organisms < or =5 microm in size dominate open ocean phytoplankton communities in terms of abundance and CO(2) fixation, with the cyanobacterial genera Prochlorococcus and Synechococcus numerically the most abundant and more extensively studied compared with small eukaryotes. However, the contribution of specific taxonomic groups to marine CO(2) fixation is still poorly known. In this study, we show that among the phytoplankton, small eukaryotes contribute significantly to CO(2) fixation (44%) because of their larger cell volume and thereby higher cell-specific CO(2) fixation rates. Within the eukaryotes, two groups, herein called Euk-A and Euk-B, were distinguished based on their flow cytometric signature. Euk-A, the most abundant group, contained cells 1.8+/-0.1 microm in size while Euk-B was the least abundant but cells were larger (2.8+/-0.2 microm). The Euk-B group comprising prymnesiophytes (73+/-13%) belonging largely to lineages with no close cultured counterparts accounted for up to 38% of the total primary production in the subtropical and tropical northeast Atlantic Ocean, suggesting a key role of this group in oceanic CO(2) fixation.

Cytometric evidence reconciling the toxicity and usefulness of CTC as a marker of bacterial activity

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 46:71-83 (2007) - doi:10.3354/ame046071 Cytometric evidence reconciling the toxicity and usefulness of CTC as a marker of bacterial activity Josep M. Gasol1,*, Javier Arístegui2 1Departament de Biologia Marina i Oceanografia, Institut de Ciències del Mar, CSIC, Pg. Marítim de la Barceloneta 37–49, 08003 Barcelona, Spain 2Facultad de Ciencias del Mar, Universidad de Las Palmas de Gran Canaria, Campus Universitario de Tafira, 35017 Las Palmas de Gran Canaria, Spain *Email: pepgasol@icm.csic.es ABSTRACT: To understand the opposing views on the utility of the CTC method, we examined bacterial abundance over incubations with the fluorogenic tetrazolium dye CTC (5-cyano-2,3-ditolyl tetrazolium chloride) and inspected the flow cytometric signatures of bacteria dually labeled with a DNA stain (Syto13) and CTC. Incubation of a marine plankton sample with CTC produced positive cells up to a stable value, reached in <1 h, while the red fluorescence of the granules increased for at least an extra hour. Incubation also produced a decrease in cell abundance of 22% after 30 min in the presence of 5 mM CTC. The decrease was a function of CTC concentration and incubation time, and particularly affected bacteria with high nucleic acid content. Flow cytometric inspection of a double-stained (CTC and Syto13) sample showed that after 15 min of incubation, particles appeared having both red (CTC+) and green (DNA+) staining. Afterwards, other particles appeared that maintained the same light scatter properties and the red fluorescence, but that lost all green fluorescence. While the number of particles with double staining stabilized after about 1 h, particles with red but without DNA staining increased for at least 100 min. Simultaneously, the classic determination of CTC+ cells (observing only the red signal of the particles) increased as reported elsewhere. We interpreted these patterns as evidence of CTF (the formazan derivative of CTC) particles growing in the bacterial cells until they are so large that they break up the cells, after which they remain present as CTF granules with no associated cellular material. Microscopic or flow cytometric enumeration of red particles might still be a good indication of the percentage of bacterial cells having taken up and reduced the activity probe, but flow cytometric cell sorting of red particles based only on scatter and red fluorescence signals will select CTF particles without associated cellular material. Our results help reconcile the ecologically sound results and the CTC toxicity evidence currently reported in the literature, and lead to a warning against interpretations of cell sorting of CTC+ particles for phylogenetic or activity studies based only on red or orange fluorescence. KEY WORDS: CTC · Syto13 · Flow cytometry · Cell sorting · Bacterial activity Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 46, No. 1. Online publication date: January 19, 2007 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2007 Inter-Research.

High-Temperature Fluorescent In Situ Hybridization for Detecting Escherichia coli in Seawater Samples, Using rRNA-Targeted Oligonucleotide Probes and Flow Cytometry

Fluorescence in situ hybridization (FISH) is a widely used method to detect environmental microorganisms. The standard protocol is typically conducted at a temperature of 46 degrees C and a hybridization time of 2 or 3 h, using the fluorescence signal intensity as the sole parameter to evaluate the performance of FISH. This paper reports our results for optimizing the conditions of FISH using rRNA-targeted oligonucleotide probes and flow cytometry and the application of these protocols to the detection of Escherichia coli in seawater spiked with E.coli culture. We obtained two types of optimized protocols for FISH, which showed rapid results with a hybridization time of less than 30 min, with performance equivalent to or better than the standard protocol in terms of the fluorescence signal intensity and the FISH hybridization efficiency (i.e., the percentage of hybridized cells giving satisfactory fluorescence intensity): (i) one-step FISH (hybridization is conducted at 60 to 75 degrees C for 30 min) and (ii) two-step FISH (pretreatment in a 90 degrees C water bath for 5 min and a hybridizing step at 50 to 55 degrees C for 15 to 20 min). We also found that satisfactory fluorescence signal intensity does not necessarily guarantee satisfactory hybridization efficiency and the tightness of the targeted population when analyzed with a flow cytometer. We subsequently successfully applied the optimized protocols to E. coli-spiked seawater samples, i.e., obtained flow cytometric signatures where the E. coli population was well separated from other particles carrying fluorescence from nonspecific binding to probes or from autofluorescence, and had a good recovery rate of the spiked E. coli cells (90%).

Different Marine Heterotrophic Nanoflagellates Affect Differentially the Composition of Enriched Bacterial Communities

We studied the effects of predation on the cytometric and phylogenetic features of two enriched bacterial communities obtained from two cultures of marine heterotrophic nanoflagellates: Jakoba libera and a mixed culture of Cafeteria sp. and Monosiga sp. Protists were harvested by flow cytometric cell sorting and eight different treatments were prepared. Each bacterial community was incubated with and without protists, and we added two treatments with protists and the bacteria present after the sorting procedure (cosorted bacteria). The bacterial community derived from the culture of Jakoba libera had higher green fluorescence per cell (FL1) than that derived from the mixed culture of Cafeteria sp. and Monosiga sp. When the experiment began all treatments presented bacterial communities that increase in fluorescence per bacterium (FL1); after that the FL1 decreased when bacteria attained maximal concentrations; and, finally, there was a new increase in FL1 toward the end of the experiment. Cosorted bacteria of Jakoba libera had the same fluorescence as the bacterial community derived from this protist, while the bacteria derived from the mixed culture of Cafeteria sp. and Monosiga sp. was nearly twice as fluorescent than that of the parental community. All treatments presented a general decline of SSC along the incubation. Therefore, there was a small influence of protists on the cytometric signature of each bacterial community. However, each bacterial community preyed by Jakoba libera or the mixed culture of Cafeteria sp. and Monosiga sp. led to four different phylogenetic fingerprint. Besides, the final Communities were different from the fingerprint of controls without protists, and most of them diverge from the fingerprint of cosorted bacteria. Our results confirm that changes in the phylogenetic composition of marine bacterial communities may depend on the initial communities of both bacteria and protists.

A proposal for optical diagnostics through the enhancement of diffraction patterns using thin-film interference filters

Coarse clumping of solid materials within diseased biological cells can have a marked influence on the light scattering pattern. Perturbations in refractive index lead to distinct variations in the cytometric signature, especially apparent over wide scattering angles. The large dynamic range of scattering intensities restricts collection of data to narrow angular intervals believed to have the highest potential for medical diagnosis. We propose the use of an interference filter to reduce the dynamic range. Selective attenuation of scattering intensity levels is expected to allow simultaneous data collection over a wide angular interval. The calculated angular transmittance of a commercial shortwave-pass filter of cut-off wavelength 580 nm indicates significant attenuation of scattering peaks below ∼10°, and reasonable peak equalization at higher angles. For the three-dimensional calculation of laser light scattered by cells we use a spectral method code that models cells as spatially varying dielectrics, stationary in time. However, we perform preliminary experimental testing with the interference filter on polystyrene microspheres instead of biological cells. A microfluidic toolkit is used for the manipulation of the microspheres. The paper intends to illustrate the principle of a light scattering detection system incorporating an interference filter for selective attenuation of scattering peaks.

Monitoring of active but non-culturable bacterial cells by flow cytometry

Flow cytometric signatures (i.e., light scatter, red and green fluorescence) were obtained for the active but non-culturable (ABNC) cells of E. coli and a coliform isolate H03N1, in seawater microcosms using BacLight, a live-dead assay kit from Molecular Probes (Eugene/Portland, OR). Previous studies have reported that there are two major adaptations, which cells undergo during the formation of ABNC states: cell wall toughening and DNA condensation. Therefore, we hypothesized that the matured ABNC forms should be more resistant to extreme temperature treatments (i.e., by freezing in liquid nitrogen and thawing at room temperature) than the normal and transition populations. It was shown that the membrane-compromised cells (comprising of normal wild-type and dead cells which are less resistant to rapid freeze thaw) could be differentiated from the matured ABNC using BacLight staining and fluorescence detection by flow cytometry. The population of ABNC cells, which could not be cultured using m-FC media (for the enumeration of fecal coliforms), was resuscitated in phosphate buffer saline followed by growth in Luria broth. Flow cytometry was thus able to detect and differentiate the ABNC cells against a mixed population comprising of culturable cells, transition populations, and dead cells. The results also showed that the formation of ABNC is as early as 2 days in seawater microcosms. By directly comparing the coliform levels enumerated by the BacLight based flow cytometry assays and m-FC technique, it was shown that the presence of coliforms can be undetected by the membrane filtration method.

Discrimination of Two Phycoerythrin-Pigment Types of Synechococcus and Their Seasonal Succession in the Uwa Sea

We identified phycoerythrin-pigment phenotypes of Synechococcus strains in samples obtained from the Uwa Sea, and investigated the seasonal changes in the pigment-type composition of the Synechococcus community in this sea from January 2001 to March 2002. Two Synechococcus strains (UT01 and UT02) with different flow cytometric signatures were isolated by sorting and subsequent culturing. Excitation spectra showed that these two strains possessed different ratios of phycourobilin (PUB) to phycoerythrobilin (PEB). Flow cytometric signatures of the two types were also different; UT01 showed a higher green to red fluorescence ratio than UT02. The ranges of green to red fluorescence intensity of the strains did not overlap, though the strains were cultured under various light intensities (7-160 μE m-2 s-1). In seawater samples, we could divide Synechococcus into lower-and higher-PUB types based on the ratio. There were two peaks of total Synechococcus abundance in June (2.8×105 cells ml-1) and August (1.6×105 cells ml-1) in the upper layer at a depth of 0 to 15 m. Major types during these two peaks were different; the higher-PUB type contributed 75.6% to the total Synechococcus community in June, and the lower-PUB type, 58.6% in August. This is the first report which describes the seasonal succession of two phycoerythrin-pigment types of Synechococcus in a coastal sea.

PICOPHYTOPLANKTON SEASONAL CYCLE AT THE SIO PIER, LA JOLLA, CALIFORNIA

The abundance of phycoerythrin‐containing cyanobacteria and picoeukaryotes in water samples from the Scripps Institution of Oceanography pier have been followed at least weekly for more than two years using flow cytometry. These cyanobacteria show a seasonal cycle with generally lower cell numbers during the winter, a “bloom” as water temperatures increase, and higher cell numbers during the summer. However the population abundance appears to be more variable and the magnitude of the annual change in cell number is less than reported for coastal Massachusetts by Waterbury et al. (1986). Isolates have been obtained from pier samples and genetic characterization using rpoC1 (RNA polymerase) sequence data is in progress. The PUB:PEB chromophore ratios of isolates assayed using fluorescence excitation spectra range from about 0.4 (low PUB) to 0.7 (mid‐PUB) for isolates grown under white light. The physiological and genetic characterization of isolates is being used to examine the similarities and differences of cyanobacterial populations from different coastal regimes. Similarly a picoeukaryote has been isolated that has a flow cytometric signature similar to the natural population. It appears to be a small nonmotile prasinophyte.

Niche-partitioning of Prochlorococcus populations in a stratified water column in the eastern North Atlantic Ocean.

The in situ community structure of Prochlorococcus populations in the eastern North Atlantic Ocean was examined by analysis of Prochlorococcus 16S rDNA sequences with three independent approaches: cloning and sequencing, hybridization to specific oligonucleotide probes, and denaturing gradient gel electrophoresis (DGGE). The hybridization of high-light (HL) and low-light (LL) Prochlorococcus genotype-specific probes to two depth profiles of PCR-amplified 16S rDNA sequences revealed that in these two stratified water columns, an obvious niche-partitioning of Prochlorococcus genotypes occurred. In each water column a shift from the HL to the LL genotype was observed, a transition correlating with the depth of the surface mixed layer (SML). Only the HL genotype was found in the SML in each water column, whereas the LL genotype was distributed below the SML. The range of in situ irradiance to which each genotype was subjected within these distinct niches was consistent with growth irradiance studies of cultured HL- and LL-adapted Prochlorococcus strains. DGGE analysis and the sequencing of Prochlorococcus 16S rDNA clones were in full agreement with the genotype-specific oligonucleotide probe hybridization data. These observations of a partitioning of Prochlorococcus genotypes in a stratified water column provide a genetic basis for the dim and bright Prochlorococcus populations observed in flow cytometric signatures in several oceanic provinces.

Cytometric diversity in marine ultraphytoplankton

The concept and methods of ecological diversity in communities were applied to phytoplankton categorized by flow cytometric measurements related to size and chlorophyll content. Each cytometric signature was condensed to single numerical values indicative of diversity and evenness. Measurements pooled from studies disparate in temporal and spatial scales indicated greater chlorophyll biomass and primary production with greater cytometric diversity and evenness. Future development of these ideas may help link biological oceanographic processes with patterns established through ecological processes at the community level.

Discrimination of marine phytoplankton species through the statistical analysis of their flow cytometric signatures

Discrimination of marine phytoplankton species through the statistical analysis of their flow cytometric signatures

A new marine picoeucaryote: Ostreococcus tauri gen. et sp. nov. (Chlorophyta, Prasinophyceae)

A new picoplanktonic alga, Ostreococcus tauri Courties et Chrétiennot-Dinet, gen. et sp. nov. (Chlorophyta, Prasinophyceae) is described from the Thau Lagoon on the Mediterranean coast, France. Almost undetectable by light or fluorescence microscopy in field studies, the cells were discovered by their flow cytometric signature and appeared numerically as the main component of the phytoplankton. Their ultrastructure is described, with additional information on cell size, pigment and DNA content. Each cell contains a nucleus, a chloroplast, one mitochondrion, one Golgi body and a very reduced cytoplasmic compartment. A starch granule and Chi a and b demonstrate its affinity to the Chlorophyta and the presence of a Chi c-like pigment, Mg 3,8 DVP (=Mg 2,4 DVP) argues for placing it in the Prasinophyceae. It differs from other coccoid taxa in ultrastructural details, mode of division and detailed pigment composition. The size and DNA content make Ostreococcus tauri the smallest eucaryote known.