Douglas S. Taylor

Nondestructive Identification of Individual Leukemia Cells by Laser Trapping Raman Spectroscopy

Currently, a combination of technologies is typically required to assess the malignancy of cancer cells. These methods often lack the specificity and sensitivity necessary for early, accurate diagnosis. Here we demonstrate using clinical samples the application of laser trapping Raman spectroscopy as a novel approach that provides intrinsic biochemical markers for the noninvasive detection of individual cancer cells. The Raman spectra of live, hematopoietic cells provide reliable molecular fingerprints that reflect their biochemical composition and biology. Populations of normal T and B lymphocytes from four healthy individuals and cells from three leukemia patients were analyzed, and multiple intrinsic Raman markers associated with DNA and protein vibrational modes have been identified that exhibit excellent discriminating power for cancer cell identification. A combination of two multivariate statistical methods, principal component analysis (PCA) and linear discriminant analysis (LDA), was used to confirm the significance of these markers for identifying cancer cells and classifying the data. The results indicate that, on average, 95% of the normal cells and 90% of the patient cells were accurately classified into their respective cell types. We also provide evidence that these markers are unique to cancer cells and not purely a function of differences in their cellular activation.

Effect of Cefazolin Treatment on the Nonresonant Raman Signatures of the Metabolic State of Individual Escherichia coli Cells

Laser tweezers Raman spectroscopy (LTRS) was used to characterize the Raman fingerprints of the metabolic states of Escherichia coli (E. coli) cells and to determine the spectral changes associated with cellular response to the antibiotic Cefazolin. The Raman spectra of E. coli cells sampled at different time points in the bacterial growth curve exhibited several spectral features that enabled direct identification of the growth phase of the bacteria. Four groups of Raman peaks were identified based on similarities in the time-dependent behavior of their intensities over the course of the growth curve. These groupings were also consistent with the different biochemical species represented by the Raman peaks. Raman peaks associated with DNA and RNA displayed a decrease in intensity over time, while protein-specific Raman vibrations increased at different rates. The adenine ring-breathing mode at 729 and the 1245 cm(-1) vibration peaked in intensity within the first 10 h and decreased afterward. Application of principal component analysis (PCA) to the Raman spectra enabled accurate identification of the different metabolic states of the bacterial cells. The Raman spectra of cells exposed to Cefazolin at the end of log phase exhibited a different behavior. The 729 and 1245 cm(-1) Raman peaks showed a slight decrease in intensity from 4 to 10 h after inoculation. Moreover, a shift in the spectral position of the adenine ring-breathing mode from 724 to 729 cm(-1), which was observed during normal bacterial growth, was inhibited during antibiotic drug treatment. These results suggest that potential Raman markers exist that can be used to identify E. coli cell response to antibiotic drug treatment.

The effect of cell fixation on the discrimination of normal and leukemia cells with laser tweezers Raman spectroscopy

Laser tweezers Raman spectroscopy (LTRS) was used to characterize the effect of different chemical fixation procedures on the Raman spectra of normal and leukemia cells. Individual unfixed, paraformaldehyde‐fixed, and methanol‐fixed normal and transformed lymphocytes from three different cell lines were analyzed with LTRS. When compared to the spectra of unfixed cells, the fixed cell spectra show clear, reproducible changes in the intensity of specific Raman markers commonly assigned to DNA, RNA, protein, and lipid vibrations (e.g. 785, 1230, 1305, 1660 cm−1) in mammalian cells, many of which are important markers that have been used to discriminate between normal and cancer lymphocytes. Statistical analyses of the Raman data and classification using principal component analysis and linear discriminant analysis indicate that methanol fixation induces a greater change in the Raman spectra than paraformaldehyde. In addition, we demonstrate that the spectral changes as a result of the fixation process have an adverse effect on the accurate Raman discrimination of the normal and cancer cells. The spectral artifacts created by the use of fixatives indicate that the method of cell preparation is an important parameter to consider when applying Raman spectroscopy to characterize, image, or differentiate between different fixed cell samples to avoid potential misinterpretation of the data.

Detection of doxorubicin-induced apoptosis of leukemic T-lymphocytes by laser tweezers Raman spectroscopy

Laser tweezers Raman spectroscopy (LTRS) was used to acquire the Raman spectra of leukemic T lymphocytes exposed to the chemotherapy drug doxorubicin at different time points over 72 hours. Changes observed in the Raman spectra were dependent on drug exposure time and concentration. The sequence of spectral changes includes an intensity increase in lipid Raman peaks, followed by an intensity increase in DNA Raman peaks, and finally changes in DNA and protein (phenylalanine) Raman vibrations. These Raman signatures are consistent with vesicle formation, cell membrane blebbing, chromatin condensation, and the cytoplasm of dead cells during the different stages of drug-induced apoptosis. These results suggest the potential of LTRS as a real-time single cell tool for monitoring apoptosis, evaluating the efficacy of chemotherapeutic treatments, or pharmaceutical testing.

Evaluation of Escherichia coli Cell Response to Antibiotic Treatment by Use of Raman Spectroscopy with Laser Tweezers

ABSTRACT Laser tweezers Raman spectroscopy was used to detect the cellular response of Escherichia coli cells to penicillin G-streptomycin and cefazolin. Time-dependent intensity changes of several Raman peaks at 729, 1,245, and 1,660 cm−1 enabled untreated cells and cells treated with the different antibiotic drugs to be distinguished.

Raman spectroscopy of individual monocytes reveals that single-beam optical trapping of mononuclear cells occurs by their nucleus

We show that laser-tweezers Raman spectroscopy of eukaryotic cells with a significantly larger diameter than the tight focus of a single beam laser trap leads to optical trapping of the cell by its optically densest part, i.e. typically the cells nucleus. Raman spectra of individual optically trapped monocytes are compared with location-specific Raman spectra of monocytes adhered to a substrate. When the cells nucleus is stained with a fluorescent live cell stain, the Raman spectrum of the DNA-specific stain is observed only in the nucleus of individual monocytes. Optically trapped monocytes display the same behavior. We also show that the Raman spectra of individual monocytes exhibit the characteristic Raman signature of cells that have not yet fully differentiated and that individual primary monocytes can be distinguished from transformed monocytes based on their Raman spectra. This work provides further evidence that laser tweezers Raman spectroscopy of individual cells provides meaningful biochemical information in an entirely nondestructive fashion that permits discerning differences between cell types and cellular activity.

Parallel analysis of individual biological cells using multifocal laser tweezers Raman spectroscopy

We report on the development and characterization of a multifocal laser tweezers Raman spectroscopy (M-LTRS) technique for parallel Raman spectral acquisition of individual biological cells. Using a 785-nm diode laser and a time-sharing laser trapping scheme, multiple laser foci are generated to optically trap single polystyrene beads and suspension cells in a linear pattern. Raman signals from the trapped objects are simultaneously projected through the slit of a spectrometer and spatially resolved on a charge-coupled device (CCD) detector with minimal signal crosstalk between neighboring cells. By improving the rate of single-cell analysis, M-LTRS is expected to be a valuable method for studying single-cell dynamics of cell populations and for the development of high-throughput Raman based cytometers.

Impact of Conditioning Regimen in Allogeneic Hematopoetic Stem Cell Transplantation for Children with Acute Myelogenous Leukemia beyond First Complete Remission: A Pediatric Blood and Marrow Transplant Consortium (PBMTC) Study

Total body irradiation (TBI)-based conditioning regimens for pediatric patients with acute myelogenous leukemia (AML) beyond first complete remission (CR1) are controversial. Because the long-term morbidity of busulfan (Bu)-based regimens appears to be lower, determining efficacy is critical. We retrospectively evaluated 151 pediatric patients with AML beyond CR1, comparing outcomes in 90 patients who received a TBI-based conditioning regimen and 61 patients who received a Bu-based conditioning regimen. There were no differences between the 2 groups with respect to age, sex, duration of CR1, time from most recent remission to transplantation, or donor source. The probability of relapse at 2 years also did not differ between the 2 groups (26% and 27%, respectively; P=.93). No significant difference in event-free survival (EFS) (P=.29) or overall survival (OS) (P=.11) was noted between the 2 groups. These findings were supported by a multivariate analysis in which TBI was not associated with improved EFS (hazard ratio [HR]=1.17; 95% confidence interval [CI]=0.66-2.10; P=.58) or OS (HR=1.42; 95% CI=0.76-2.64; P=.27). Shorter CR1 and receiving an HLA-mismatched transplant adversely affected EFS and OS in this cohort. Our study provides no evidence of an advantage to using TBI in children with AML beyond CR1. A prospective, randomized study is needed to confirm these results.

Advancement of Pediatric Blood and Marrow Transplantation Research in North America: Priorities of the Pediatric Blood and Marrow Transplant Consortium

Advances in pediatric bone marrow transplantation (BMT) are slowed by the small number of patients with a given disease who undergo transplantation, a lack of sufficient infrastructure to run early-phase oncology protocols and studies of rare nonmalignant disorders, and challenges associated with funding multi-institutional trials. Leadership of the Pediatric Blood and Marrow Transplant Consortium (PBMTC), a large pediatric BMT clinical trials network representing 77 active and 45 affiliated centers worldwide, met in April 2009 to develop strategic plans to address these issues. Key barriers, including infrastructure development and funding, along with scientific initiatives in malignant and nonmalignant disorders, cellular therapeutics, graft-versus-host disease, and supportive care were discussed. The PBMTCs agenda for approaching these issues will result in infrastructure and trials specific to pediatrics that will run through the PBMTC or its partners, the Blood and Marrow Transplant Clinical Trials Network and the Childrens Oncology Group.

Coagulation profile of liquid-state plasma.

BACKGROUND: Use of liquid plasma (LP) has been reported as early as the mid 1930s. Unlike fresh‐frozen plasma (FFP), LP is maintained at 1 to 6°C for up to 40 days after collection and processing. Despite its approved use by the US Food and Drug Administration, the coagulation profile of LP is incompletely described. In this study we evaluate the coagulation profile of LP stored up to 30 days.