isolation-primary-human-colon-tumor-cells-from-surgical-tissues

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

University of Illinois at Urbana-Champaign

Provena Covenant Medical Centre

To present a case of cellulitis/myositis due to Stenotrophomonas maltophilia in the absence of trauma and to discuss a potentially novel treatment option.

Severe cellulitis/myositis caused by Stenotrophomonas maltophilia.

Muir-Torre syndrome (MTS) is a rare disorder characterized by the presence of at least one sebaceous gland neoplasm and at least one visceral malignancy. Sebaceous adenomas, sebaceous carcinomas, and sebaceomas (sebaceous epitheliomas) are all characteristic glandular tumors of MTS. The most common visceral malignancies associated with MTS are colorectal, followed by genitourinary. These visceral malignancies frequently have a more indolent course in patients with MTS than they would otherwise. Muir-Torre syndrome is an autosomal dominant disorder; however, sporadic cases are known to develop. It often is associated with germ-line mutations in the mutS homolog 2, colon cancer, nonpolyposis type 1 (Escherichia coli) gene, MSH2, and the mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli) gene, MLH1 (similar to hereditary nonpolyposis colon cancer [HNPCC]). The diagnosis of MTS currently is based on clinical criteria; however, immunohistochemical staining for MSH2 and MLH1 can confirm the diagnosis. We report 2 patients with MTS who developed colon adenocarcinomas in conjunction with sebaceous carcinomas. Both patients demonstrated loss of MSH2 expression in tumor cells on immunohistochemical staining. One of these patients later developed gastric carcinoma, a very uncommon malignancy associated with MTS. We conclude that the diagnosis of rare sebaceous lesions associated with MTS may represent a marker of visceral disease and warrants further investigation for internal malignancies in the individual and at-risk family members.

Muir-Torre syndrome: a rare but important disorder.

We demonstrate Diffraction Phase Cytometry (DPC) as a single shot, full-field, high throughput quantitative phase imaging modality, dedicated to analyzing whole blood smears. Utilizing a commercial CD as a sample substrate, along with dynamic spatial filtering via a liquid crystal spatial light modulator, we have developed a compact instrument capable of making quantitative, physiologically relevant measurements. To illustrate the ability of the system to function as a highly sensitive cytometer we imaged a large number (N=1,537) of live human erythrocytes in whole blood without preparation. We retrieved a comprehensive set of geometrical parameters including cell volume and surface area, which are not directly available using existing cytometers. Furthermore, we retrieved the minimum cylindrical diameter, through which red blood cells can pass, and deliver oxygen. These initial results prove the concept for an inexpensive lab-on-a-chip blood screening device.

Diffraction Phase Cytometry: blood on a CD-ROM.

Blood smear analysis has remained a crucial diagnostic tool for pathologists despite the advent of automatic analyzers such as flow cytometers and impedance counters. Though these current methods have proven to be indispensible tools for physicians and researchers alike, they provide limited information on the detailed morphology of individual cells, and merely alert the operator to manually examine a blood smear by raising flags when abnormalities are detected. We demonstrate an automatic interferometry-based smear analysis technique known as diffraction phase cytometry (DPC), which is capable of providing the same information on red blood cells as is provided by current clinical analyzers, while rendering additional, currently unavailable parameters on the 2-D and 3-D morphology of individual red blood cells. To validate the utility of our technique in a clinical setting, we present a comparison between tests generated from 32 patients by a state of the art clinical impedance counter and DPC.

Blood screening using diffraction phase cytometry.

Confocal Raman microscopy is often used for optical sectioning but is problematic when the sample plane of interest has a weak Raman cross-section/signal relative to areas that are out-of-focus. This is especially true for clinical samples in pathology, which consist of a thin tissue (approximately 5 microm) sample placed on a thick glass slide. Here, we recognize that the problem is the result of the extent of the illumination at the confocal plane being larger than the size of the sample and propose a dark field illumination scheme to efficiently reject substrate signals. The ability of several optical configurations in rejecting out-of-plane signal is investigated for two model systems: SU-8 photo resist over Teflon and SU-8 photo resist over polystyrene. The proposed reflective dark field approach, in which excitation converged to a focal point slightly above the focal plane of the collection optics, was found to be most effective in recording data from the sample. The proposed approach is validated by the rejection of substrate response (fluorescence) in spectra acquired from approximately 4 microm of breast tissue on a glass microscope slide. The proposed approach is easy to implement on existing confocal systems, has a straightforward optimization in acquiring data, and is not expected to result in loss of lateral resolution in mapping experiments.

Dark field Raman microscopy.

The leading cause of line-of-duty death among firefighters is sudden cardiac events. Platelets play a critical role in the formation of an occlusive thrombus during an ischemic event.

Effect of live-fire training drills on firefighters' platelet number and function.

La principale cause de d&eacute;c&egrave;s de line-of-duty des pompiers est &eacute;v&eacute;nements cardiaques soudains. Les plaquettes jouent un r&ocirc;le essentiel dans la formation d&#39;un thrombus occlusif pendant un &eacute;v&eacute;nement isch&eacute;mique.

Effet des exercices d&#39;entra&icirc;nement au tir r&eacute;el sur le nombre de plaquettes et la fonction des pompiers.

La principale causa di morte di linea-di-dovere tra i vigili del fuoco &egrave; eventi cardiaci improvvisi. Le piastrine svolgono un ruolo fondamentale nella formazione di un trombo occlusivo durante un evento ischemico.

Effetto del fuoco vivo formazione Trapani sul numero delle piastrine e la funzione dei vigili del fuoco.

We present optical measurements of nanoscale red blood cell fluctuations obtained by highly sensitive quantitative phase imaging. These spatio-temporal fluctuations are modeled in terms of the bulk viscoelastic response of the cell. Relating the displacement distribution to the storage and loss moduli of the bulk has the advantage of incorporating all geometric and cortical effects into a single effective medium behavior. The results on normal cells indicate that the viscous modulus is much larger than the elastic one throughout the entire frequency range covered by the measurement, indicating fluid behavior.

Effective 3D viscoelasticity of red blood cells measured by diffraction phase microscopy.

Tumor-induced osteomalacia is a rare acquired condition characterized by phosphaturia, hypophosphatemia and osteomalacia. We report an unusual presentation in a 15-year-old healthy male with a two-week history of cough and chest pain. The chest radiograph showed right middle lobe opacity and chest CT revealed a mass in the extra pleural space. A biopsy showed chondro-myxoidstroma with osteoid formation. Diagnosis was confirmed with the above findings and hypophosphatemia. The patient's symptoms resolved after complete surgical excision of the mass. Tumor-induced osteomalacia, although a rare disorder, can be a diagnostic challenge, especially in patients presenting with atypical symptoms.

Tumor-induced osteomalacia (TIO): atypical presentation.

The primary role of a red blood cell (RBC) is delivering oxygen throughout our body. Abnormalities of this basic function lead to anemia and are caused by numerous diseases such as malaria and sickle cell anemia. As prompt and inexpensive tests for blood screening are in demand, we have developed a faster and reliable way to measure morphological parameters associated with the structure of red blood cells and the size distribution of the cells in a whole blood smear. Modeling the RBC shape under Born approximation, we are able to determine parameters of clinical relevance, such as the diameter, thickness and dimple size. From a measured quantitative phase image of a blood smear, we can determine the average and standard deviation of the red blood cell volume simultaneously, i.e., without analyzing each cell individually. This approach may open the door for a new generation of label-free, high-throughput blood testing.

Born approximation model for light scattering by red blood cells.

We report experimental evidence of correlation-induced spectral changes in biological tissues. The overall spectral shift in our transmission measurements is to the red and the mean wavelength of the original spectrum is up 10% larger. These results indicate that the spectral changes due to elastic scattering are significant and likely to hinder all spectroscopic measurements based on the inelastic (i.e., emission and absorption) interaction between light and tissues. Thus, simultaneous morphology and spectral measurements are required for accurate measurements spectroscopic information.

Correlation-induced spectral changes in tissues.

The gold standard in histopathology relies on manual investigation of stained tissue biopsies. A sensitive and quantitative method for in situ tissue specimen inspection is highly desirable, as it would allow early disease diagnosis and automatic screening. Here we demonstrate that quantitative phase imaging of entire unstained biopsies has the potential to fulfill this requirement. Our data indicates that the refractive index distribution of histopathology slides, which contains information about the molecular scale organization of tissue, reveals prostate tumors and breast calcifications. These optical maps report on subtle, nanoscale morphological properties of tissues and cells that cannot be recovered by common stains, including hematoxylin and eosin. We found that cancer progression significantly alters the tissue organization, as exhibited by consistently higher refractive index variance in prostate tumors versus normal regions. Furthermore, using the quantitative phase information, we obtained the spatially resolved scattering mean free path and anisotropy factor g for entire biopsies and demonstrated their direct correlation with tumor presence. In essence, our results show that the tissue refractive index reports on the nanoscale tissue architecture and, in principle, can be used as an intrinsic marker for cancer diagnosis.

Tissue refractive index as marker of disease.

It has recently been shown that quantitative phase imaging methods can provide clinically relevant parameters for red blood cell analysis with unprecedented detail and sensitivity. Since the quantitative phase information is dependent on both the thickness and refractive index, a major limitation to clinical translation has been a simple and practical approach to measure both simultaneously. Here we demonstrate both theoretically and experimentally that, by combining quantitative phase with a single absorption measurement, it is possible to measure both quantities at the single cell level. We validate this approach by comparing our results to those acquired using a clinical blood analyzer. This approach to decouple the thickness and refractive index for red blood cells may be used with any quantitative phase imaging method that can operate in tandem with bright field microscopy at the Soret-band wavelength.

Blood testing at the single cell level using quantitative phase and amplitude microscopy.

Numerous techniques have been developed for localizing lymph nodes before surgical resection and for their histological assessment. Nondestructive high-resolution transcapsule optical imaging of lymph nodes offers the potential for in situ assessment of metastatic involvement, potentially during surgical procedures.

Three-dimensional optical coherence tomography for optical biopsy of lymph nodes and assessment of metastatic disease.

We introduce a label-free method for detecting high-grade prostatic intraepithelial neoplasia (HGPIN) in unstained biopsies. We image this condition based on the identification of basal cells in biopsies that otherwise would resemble prostate cancer by unassisted histologic examination. Gradient field microscopy (GFM) is used as a label-free imaging method which increases the contrast of a transparent sample by taking the first-order phase derivative that is very sensitive to rapid refractive index changes. GFM is able to image the basal cell layers in HGPIN biopsies because of their rapid refractive index changes at the periphery of small glandular structures.

Gradient field microscopy for label-free diagnosis of human biopsies [Invited].

We demonstrate a real-time blood testing system that can provide remote diagnosis with minimal human intervention in economically challenged areas. Our instrument combines novel advances in label-free optical imaging with parallel computing. Specifically, we use quantitative phase imaging for extracting red blood cell morphology with nanoscale sensitivity and NVIDIA's CUDA programming language to perform real time cellular-level analysis. While the blood smear is translated through focus, our system is able to segment and analyze all the cells in the one megapixel field of view, at a rate of 40 frames/s. The variety of diagnostic parameters measured from each cell (e.g., surface area, sphericity, and minimum cylindrical diameter) are currently not available with current state of the art clinical instruments. In addition, we show that our instrument correctly recovers the red blood cell volume distribution, as evidenced by the excellent agreement with the cell counter results obtained on normal patients and those with microcytic and macrocytic anemia. The final data outputted by our instrument represent arrays of numbers associated with these morphological parameters and not images. Thus, the memory necessary to store these data is of the order of kilobytes, which allows for their remote transmission via, for example, the cellular network. We envision that such a system will dramatically increase access for blood testing and furthermore, may pave the way to digital hematology.

Real time blood testing using quantitative phase imaging.

Laser-based Fourier phase microscopy (FPM) works on the principle of decomposition of an image field in two spatial components that can be controllably shifted in phase with respect to each other. However, due to the coherent illumination, the contrast in phase images is degraded by speckles. In this paper we present FPM with spatially coherent white light (wFPM), which offers high spatial phase sensitivity due to the low temporal coherence and high temporal phase stability due to common path geometry. Further, by using a fast spatial light modulator (SLM) and a fast scientific-grade complementary metal oxide semiconductor (sCMOS) camera, we report imaging at a maximum rate of 12.5 quantitative phase frames per second with 5.5 mega pixels image size. We illustrate the utility of wFPM as a contrast enhancement as well as dynamic phase measurement method by imaging section of benign colonic glands and red blood cell membrane fluctuation.

Fourier phase microscopy with white light.

Studying the dynamics of fibrin clot formation and its morphology is an important problem in biology and has significant impact for several scientific and clinical applications. We present a label-free technique based on quantitative phase imaging to address this problem. Using quantitative phase information, we characterized fibrin polymerization in real-time and present a mathematical model describing the transition from liquid to gel state. By exploiting the inherent optical sectioning capability of our instrument, we measured the three-dimensional structure of the fibrin clot. From this data, we evaluated the fractal nature of the fibrin network and extracted the fractal dimension. Our non-invasive and speckle-free approach analyzes the clotting process without the need for external contrast agents.

Spatiotemporal characterization of a fibrin clot using quantitative phase imaging.

Mastocytosis is a type of myeloproliferative neoplasm characterized by accumulation and proliferation of morphologically and immunophenotypically abnormal mast cells in 1 or more organ systems. Clinical manifestations vary depending upon the organ involved and chemical mediators released by mast cells along with constitutional symptoms and musculoskeletal complaints. We report a case of isolated bone marrow mastocytosis in an 87-year-old woman who presented with a fall resulting in proximal femur fracture. Bone marrow biopsy revealed mastocytosis, and no evidence of systemic involvement or peripheral mastocytosis was found. Physicians should be aware of this entity, especially in patients with osteoporosis.

Mastocytosis as an unusual cause of hip fracture in an elderly woman.

The risk of biochemical recurrence of prostate cancer among individuals who undergo radical prostatectomy for treatment is around 25%. Current clinical methods often fail at successfully predicting recurrence among patients at intermediate risk for recurrence. We used a label-free method, spatial light interference microscopy, to perform localized measurements of light scattering in prostatectomy tissue microarrays. We show, for the first time to our knowledge, that anisotropy of light scattering in the stroma immediately adjoining cancerous glands can be used to identify patients at higher risk for recurrence. The data show that lower value of anisotropy corresponds to a higher risk for recurrence, meaning that the stroma adjoining the glands of recurrent patients is more fractionated than in non-recurrent patients. Our method outperformed the widely accepted clinical tool CAPRA-S in the cases we interrogated irrespective of Gleason grade, prostate-specific antigen (PSA) levels and pathological tumor-node-metastasis (pTNM) stage. These results suggest that QPI shows promise in assisting pathologists to improve prediction of prostate cancer recurrence.

Article	Year
Severe cellulitis/myositis caused by Stenotrophomonas maltophilia. The Annals of pharmacotherapy\| PubMed ID: 11816260	2002
Muir-Torre syndrome: a rare but important disorder. Cutis\| PubMed ID: 19055168	2008
Diffraction Phase Cytometry: blood on a CD-ROM. Optics express\| PubMed ID: 19219161	2009
Blood screening using diffraction phase cytometry. Journal of biomedical optics\| PubMed ID: 20459290	2010
Dark field Raman microscopy. Analytical chemistry\| PubMed ID: 20568722	2010
Effect of live-fire training drills on firefighters' platelet number and function. Prehospital emergency care : official journal of the National Association of EMS Physicians and the National Association of State EMS Directors\| PubMed ID: 21366433	2011
Effective 3D viscoelasticity of red blood cells measured by diffraction phase microscopy. Biomedical optics express\| PubMed ID: 21412454	2011
Tumor-induced osteomalacia (TIO): atypical presentation. Southern medical journal\| PubMed ID: 21606715	2011
Born approximation model for light scattering by red blood cells. Biomedical optics express\| PubMed ID: 22025984	2011
Correlation-induced spectral changes in tissues. Optics letters\| PubMed ID: 22048367	2011
Tissue refractive index as marker of disease. Journal of biomedical optics\| PubMed ID: 22112122	2011
Blood testing at the single cell level using quantitative phase and amplitude microscopy. Biomedical optics express\| PubMed ID: 22162816	2011
Three-dimensional optical coherence tomography for optical biopsy of lymph nodes and assessment of metastatic disease. Annals of surgical oncology\| PubMed ID: 22688663	2013
Gradient field microscopy for label-free diagnosis of human biopsies [Invited]. Applied optics\| PubMed ID: 23292427	2013
Real time blood testing using quantitative phase imaging. PloS one\| PubMed ID: 23405194	2013
Fourier phase microscopy with white light. Biomedical optics express\| PubMed ID: 24010005	2013
Spatiotemporal characterization of a fibrin clot using quantitative phase imaging. PloS one\| PubMed ID: 25386701	2014
Mastocytosis as an unusual cause of hip fracture in an elderly woman. WMJ : official publication of the State Medical Society of Wisconsin\| PubMed ID: 25745700	2014
Prediction of prostate cancer recurrence using quantitative phase imaging. Scientific reports\| PubMed ID: 25975368	2015

Krishnarao Tangella

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