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Validation of Large Particle Flow Cytometry for the Analysis and Sorting of Intact Pancreatic Islets

Fernandez, Luis A.1; Hatch, Eric W.1; Armann, Barbara2; Odorico, Jon S.1; Hullett, Debra A.1; Sollinger, Hans W.1; Hanson, Matthew S.1

doi: 10.1097/01.tp.0000179105.95770.cd
Original Articles: Cell Therapy and Islet Transplantation
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Background. Accurate quantification of total islet yield is an essential step prior to transplantation and for research. The standard method of manually determining an islet equivalent (IEQ) count is subjective and prone to error. We evaluated Complex Object Parametric Analyzer and Sorter (COPAS) large particle flow cytometry for the determination of islet equivalent counts and purities of islet preparations.

Methods. Initial validation of the sensitivity and accuracy of the COPAS flow cytometer was performed by analysis and sorting of uniform polystyrene microspheres with sizes similar to islets. Human and Rhesus monkey islets were stained with the zinc-specific fluorescent dye Newport Green to discriminate islet from nonislet tissue. Islet sizes were extrapolated from standard curves obtained using microspheres from which individual islet volumes were calculated. IEQ counts on six islet preparations were performed by the standard manual method and compared with results obtained by automated COPAS flow cytometry.

Results. The COPAS flow cytometer was highly accurate in the detection and measurement of both polystyrene microspheres and islets. IEQ counts determined by COPAS flow cytometry were consistent with manual counts although subject to error when assessing preparations with significant numbers of islets embedded within acinar tissue. Size-specific islet sorting with retention of morphology and dithizone staining was also shown using the COPAS flow cytometer.

Conclusions. COPAS large particle flow cytometry provides a novel automated approach for quantification of intact islets and determination of islet equivalent yield. In addition, the ability to analyze and sort islets based upon user defined criterion opens unique avenues for experimentation.

1Department of Surgery, University of Wisconsin-Madison, Madison, WI.

2Department of Surgery, University of Leipzig, Germany.

This work was supported by internal funds from the Division of Transplantation at the University of Wisconsin-Madison Medical School.

Address correspondence to: Luis A. Fernandez, M.D., Department of Surgery, University of Wisconsin-Madison, UW Hospitals & Clinics, H5/301, 600 Highland Ave., Madison, WI 53792-3236.

E-mail: luisf@surgery.wisc.edu.

Received 12 November 2004. Revision requested 15 December 2004.

Accepted 6 April 2005.

Successful replication of the Edmonton islet transplantation protocol at a diversity of large and small institutions world-wide has established its efficacy as a therapy for patients with type 1 diabetes (1). Although islet isolation and transplantation procedures have become more standardized, the methods of quantifying the final purified islet yield still lack the precision and reproducibility desired of such a critical step. Typically, a small sample of the final islet preparation is stained with dithizone and observed using an inverted microscope. The approximate diameters of the islets are determined manually by the reader using an eyepiece reticle and normalized to the volume of a 150-μm diameter sphere. This calculation is based upon the islet equivalent (IEQ) as the standard unit of islet volume as adopted by the Second International Congress on Pancreas and Islet Transplantation (2). Despite the speed and ease of this method of islet enumeration it is prone to error due to intra- and inter-reader variability, compounding the already difficult procedure of obtaining a representative sample. Alternative methods using computerized image analysis, sedimentation, DNA quantification, or electrozone sensing have all been attempted with varying levels of success, but no automated method has become a standard (3–6). A reproducible method is desired in which human error can be subtracted to maximize the accuracy of the IEQ determination.

Flow cytometers are well-established research and clinical instruments that can provide rapid, quantifiable, and verifiable data on cellular phenotype, differentiation, and metabolic state. Union Biometrica’s COPAS instruments, which stand for Complex Object Parametric Analyzer and Sorter, can analyze and sort objects ranging from 40 μm to 1.5 mm in diameter. Three main differences between the COPAS and other flow cytometers are the size of the objects that can be analyzed, the amount of shear force that these objects are subjected to, and the method of sorting and dispensing. All three of these factors make it difficult to run islets on conventional flow cytometers designed for single cells. The COPAS has the ability to run large objects with minimal shear forces, and to sort these objects with a mechanism that is gentle enough to dispense living organisms without affecting viability. The current COPAS instruments can quickly and accurately analyze and sort individual objects based on five parameters: size, optical density, and up to three spectra of fluorescence. Each of these parameters can be observed and recorded by computer for each object that passes individually through the instrument. The purpose of this study was to validate the capabilities of COPAS flow cytometry as an automated method for quality control assessments of intact islets.

An essential tool required for the analysis and quantification of islets by flow cytometry is a marker to discriminate islets from nonislet particles. Newport Green (NG) is a fluorescent liposoluble diacetate ester with binding affinity for Zinc (Zn2+) that has been reported to show beta cell specific staining (7). Pancreatic beta cells contain large concentrations of Zn2+, which is involved in the synthesis, storage, and secretion of insulin. We evaluated the specificity of Newport Green staining on intact human and Rhesus monkey islets when analyzed and sorted by COPAS flow cytometry. Our data show that COPAS analysis and sorting of Newport Green stained islets is a reliable automated method for the determination of islet equivalent counts and could potentially represent a novel quality control instrument beneficial to the fields of islet transplantation and research.

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MATERIALS AND METHODS

Monosized Polystyrene Microspheres

Uniform polystyrene microspheres (fluorescent and nonfluorescent) were purchased from Duke Scientific Corporation (Palo Alto, CA) and suspended in deionized water containing 0.1% Triton X-100. Microsphere diameters were certified by the manufacturer using photon correlation spectroscopy, transmission electron microscopy, or optical microscopy. Microspheres were diluted in phosphate buffered saline (PBS without Ca2+ or Mg2+) for analysis by COPAS flow cytometry (Union Biometrica, Boston, MA). Initial quantification of beads placed in solution was accomplished using manufacturer supplied formulas for the number of beads of each diameter expected per gram dry weight. Confirmation of bead sample concentrations was accomplished manually by counting aliquots using an inverted microscope with eyepiece reticle. A mock islet sample was prepared containing a total of 10,246 microspheres (1325× 41.5 μm, 1327× 109 μm, 3000× 161 μm, 2000× 200 μm, 1699× 239 μm, 595× 298 μm, and 300× 400 μm) and their volume in islet equivalents was calculated based upon the manufacturer’s size specifications.

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Islet Purification

Human pancreata were obtained through the University of Wisconsin Hospitals and Clinics Organ Procurement Organization with consent obtained for research. Rhesus macaque pancreata were obtained from the Wisconsin National Primate Center following guidelines established by the University of Wisconsin-Madison Institutional Animal Care and Use Committee. Islets were isolated using a modification of the automated Ricordi method (8). For human islet isolations Collagenase NB1, Neutral Protease (Serva Electrophoresis, Heidelberg, Germany) and DNase (Roche Applied-Sciences, Indianapolis, IN) in Hanks’ balanced salt solution was infused into the main pancreatic duct using a handheld syringe and 16 ga angiocath. For Rhesus islet isolations Liberase CI (Roche Applied-Sciences, Indianapolis, IN) was used for pancreas digestion. The Ricordi chamber was shaken manually during the digestion phase and with a mechanical shaker during the dilution phase. Islets were separated from contaminating exocrine tissue by centrifugation on a continuous Biocoll gradient (Biochrom AG, Berlin, Germany) on a COBE 2991 cell processor (Lakewood, CO). Islets were cultured in vitro in CMRL supplemented medium (Mediatech, Herndon, VA) containing 0.5% human serum albumin at 37°C prior to experimentation.

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Newport Green Staining of Islets

Aliquots of islets cultured for 1 to 7 days were harvested from flasks and incubated in a buffer containing phosphate buffered saline (PBS, without Ca2+ or Mg2+), 10 mM HEPES, 3 mM EGTA, 2.8 mM glucose, and 2.5 mg/ml BSA for 5 min at 37°C to aid dye penetration into the islet. Islets were then washed with CMRL supplemented media containing 0.5% human serum albumin and resuspended at a concentration of approximately 300 islet particles/ml of culture media. Pluronic (Molecular Probes, Eugene, OR), a nonionic and nondenaturing detergent was added at a concentration of 1.5 μl/ml from a 20% DMSO stock solution to help solubilize the Newport Green DCF ester and enhance cell loading. Newport Green DCF (Molecular Probes, Eugene, OR) was added to a final concentration of 10 μM and islets were incubated at 37°C for 30 min. Islets were washed twice in culture media, incubated for an additional 15 min at 37°C to ensure complete de-esterification of the dye within cells, and then held on ice prior to analysis and sorting on the COPAS flow cytometer. Digital photomicroscopy was performed using a Zeiss Axiovert 200 MAT/TV inverted microscope (Carl Zeiss Microimaging, Thornwood, NY).

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Insulin Immunohistochemistry

Islets were fixed in 1% buffered formaldehyde for 60 min, resuspended in liquified HistoGel (Richard-Allan Scientific, Kalamazoo, MI) and then allowed to polymerize on ice. Encased islets were then processed for paraffin embedding. Sections of 4 μm were pretreated by the HIER method in 10 mmol/L citric acid (pH 6.0) by heating in a pressure cooker for 2 min and cooled at room temperature for 20 min. After permeabilization (0,1% Triton X-100 in PBS), endogenous peroxidase was blocked by 3% hydrogen peroxide in methanol for 10 min. Nonspecific binding was blocked with Background Sniper (Biocare Medical, Walnut Creek, CA) for 9 min. Incubation with primary antibody (guinea pig anti-pig insulin, Sigma, St. Louis, MO) was 45 min at room temperature. The peroxidase-conjugated secondary antibody was directed against guinea-pig IgG (Sigma, St. Louis, MO). The primary antibody was detected by Diaminobenzidine (R&D, Minneapolis, MN) for 3 min and the slides were counterstained with Harris Hematoxylin.

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Complex Object Parametric Analyzer and Sorter (COPAS)

The COPAS is equipped with a quartz flow cell (1 mm ID) and 670 nm red laser and 488/514 multi-line lasers. The emission of the red laser is collected by an optical detector and creates the Extinction (EXT) parameter and the Time of Flight (TOF) parameter. The EXT parameter is a measure of particle optical density and TOF is a measure of a particle’s largest axis. The emission of fluorochromes excited by the multi-line laser can be detected by three separate photon multiplier tubes (PMT) with collection wavelengths separated by dichroic mirrors (510 nm, 545 nm, and 580 nm). The sample and sheath streams are diverted after analysis to a waste collector. Sorting is accomplished based upon criteria defined in the acquisition software by switching off the diverter for a set period of time to allow the particle to be collected. Sorted particles can be dispensed into a variety of vessel sizes containing user selected buffers or media. The COPAS used in this study can accurately measure particles ranging from 40 to 500 μm in cross-sectional diameter.

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Islet Equivalent Determinations

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Manual Method

Triplicate samples containing between 100 and 300 islets were stained with dithizone (Sigma-Aldrich, St. Louis, MO) and sized using an eyepiece reticle and inverted microscope (9). All islets with a diameter ≥50 μm were divided into classes of 50 μm increments (i.e., 50–100, 100–150, 150–200, etc.) for calculation of the IEQ. Table 1a indicates the mean volume for each diameter class and the relative conversion factor into islets of 150-μm diameter. These factors make it possible to convert the total islet number from any preparation into islet equivalent (IEQ).

TABLE 1a

TABLE 1a

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Automated COPAS Flow Cytometry Method

A standard curve of particle size versus time of flight (TOF) was obtained by analysis of samples containing uniform microspheres of each of the following sizes (41.5, 109, 200, 298, and 400 μm). A second-order polynomial regression line was fit to the microsphere diameter versus mean TOF data. The diameters of particles of unknown size were extrapolated from the polynomial regression line equation using their individual TOF values and converted to volume (volume of a sphere = 4/3 π r3). The individually calculated volumes for each particle were summed and divided by the volume of a 150-μm sphere (1,767,146 μm3) to determine the total islet equivalent (IEQ) count.

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Data Analysis and Statistics

COPAS flow cytometry data were analyzed using WinMDI software. Polynomial regression analysis of data and computation of descriptive statistics was performed with Microsoft Excel and Statistica software.

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RESULTS

Measurement of Particle Size by COPAS Flow Cytometry

Prior to utilizing the COPAS flow cytometer as a tool for analyzing intact islets, it was necessary to assess the accuracy and precision by which the instrument could measure particles of similar size and volume. Polystyrene microspheres manufactured to uniform diameters ranging from 41.5 to 400 μm were analyzed by COPAS flow cytometry. Time of flight (TOF) is the parameter measuring the duration of time that an object spends in the excitation lasers path and is proportional to the objects size. As shown in Figure 1, a sample mixture of microspheres comprised of five specific sizes could be clearly resolved based upon TOF. A second order polynomial regression of the data provided the best fit line (R2 ≥ 0.99). As particle size increases the TOF value increases nonlinearly due to the parabolic nature of the sample streams flow rate within the sheath stream. These data show that a standard curve of mean TOF versus microsphere diameter can be established from which the diameters of particles of unknown size can be extrapolated individually based upon their TOF values. Comparison of a wide range of microsphere sizes measured by COPAS with the manufacturer’s specifications demonstrated the accuracy of this method (Table 1). The COPAS measurements of eight distinct microsphere sizes were all found to be within 5% of the manufacturer’s specifications.

FIGURE 1.

FIGURE 1.

TABLE 1

TABLE 1

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COPAS Sample Recovery and Sorting Assessment

To assess the potential error of COPAS measurements due to incomplete sampling, we evaluated the percentage of sample recovery. Samples containing known quantities of microspheres were analyzed on the COPAS flow cytometer and the number of data points acquired was compared with expected (Table 2). Satisfactory recovery percentages were obtained for all eight distinct microsphere sizes. The precision of particle sorting by COPAS was tested by sorting samples containing mixtures of green fluorescent microspheres of different sizes into uniform sizes (Table 3). Fluorescent microspheres were utilized to allow sorting based upon both size and fluorescence. Sample recoveries and the size-specificity of sorting were satisfactory for all microsphere sizes tested.

TABLE 2

TABLE 2

TABLE 3

TABLE 3

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COPAS Flow Cytometry Accurately Determines an Islet Equivalent Count for a Mock Islet Sample of Polystyrene Microspheres

A sample was prepared using polystyrene microspheres of known diameter and quantity to mimic the diversity of particle sizes present within an actual islet preparation (see methods). Three separate methods of determining the total islet equivalent count were then compared (Table 4). The “calculated” method utilized the manufacturer’s certified specifications for the individual microsphere diameters and the known quantity of each within the sample to calculate the total volume, which was then converted to IEQ. The “standard” method was to follow the typical method used for dithizone stained islets observed by inverted microscopy and measured by eyepiece reticle. In this method islets are grouped into 50-μm size ranges and their volumes normalized to the volume of a 150-μm sphere to determine the total IEQ in the sample. The eight different microsphere sizes were grouped into 50-μm size ranges based upon their calculated frequency within a 300 particle sample. The “COPAS” method was to analyze samples containing either 100, 300, or 600 particles on the COPAS flow cytometer, extrapolate the diameter of each microsphere individually, calculate the total volume, which was then converted to total IEQ. The total IEQ count determined by the standard method of grouping particles into 50-μm size ranges was found to be a 14.7% overestimate when compared to the IEQ count calculated using the manufacturer’s specifications. The total IEQ count determined from samples measured by the COPAS flow cytometer showed superior accuracy to the standard method when the sample size contained greater than 300 individual particles. IEQ determinations from samples containing 300 or 600 particles came within 5% of the calculated total IEQ count. The improved accuracy of the IEQ count determined by COPAS measurement with increasing sample size was likely reflective of the difficulty of obtaining a truly representative sample of particles in a small volume.

TABLE 4

TABLE 4

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Discrimination of Islets from Nonislet Tissue Utilizing Newport Green

Accurate determination of an IEQ count requires a method of discriminating islet from non-islet pancreatic tissue. Successful purification of islets by gradient centrifugation still typically yields preparations containing between 10–20% nonislet tissue. Newport Green is a cell permeant diacetate ester that has moderate binding affinity for intracellular zinc (Kd (Zn2+) 1 μM) and is insensitive to Ca2+ and Mg2+ (10). In contrast to the low intracellular concentration of zinc present in most cells, islet beta cells have a high level associated with insulin granules. The high zinc content of beta cells allows for specific staining using dithizone and has been shown by others to also work with Newport Green (7). Semipurified preparations of human and Rhesus monkey islets were stained intact with Newport Green and analyzed for fluorescence by COPAS flow cytometry. Although, unstained islet and nonislet acinar tissue showed detectable levels of background autofluorescence, the islet-specific signal obtained with Newport Green was sufficient to provide discrimination from nonislet tissue (Fig. 2). As a means of assessing the specificity of Newport Green staining for islets, particles giving signals above background were sorted and stained with dithizone. In repeated experiments 90–99% of sorted Newport Green positive particles also stained with dithizone (Table 5). Those particles not staining intensely were typically observed to show very low level or heterogenous dithizone staining upon more careful microscopic evaluation.

FIGURE 2.

FIGURE 2.

TABLE 5

TABLE 5

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Determination of an IEQ Count by Newport Green Staining and COPAS Flow Cytometry

Islet equivalent counts on samples from six separate islet preparations were determined manually using the standard dithizone and optical microscopy method and compared to counts obtained using Newport Green and COPAS flow cytometry (Table 5). In five of six comparisons, the IEQ count determined by Newport Green staining and COPAS flow cytometry were similar to the values calculated by the standard method. Moreover, the coefficients of variation (CV) of the manual and COPAS counts were not significantly different from each other. In contrast, in one experiment (exp. 2, Table 5) the COPAS count was twofold greater than the manual count. The islets used in this experiment contained a large number that were embedded or “mantled” within acinar tissue. The anomalous COPAS IEQ count reflects the fact that TOF data are representative of the entire particle size and do not discriminate islet from nonislet tissue within the same particle.

A significant advantage of the flow cytometric method of assessing islet preparations is the ability to calculate an accurate percent purity. The sizes and volumes of all Newport Green positive and negative particles in each sample were calculated by the automated COPAS method. The percent purity was calculated by dividing the total islet volume by the total volume of tissue in the sample. Comparison of purity measurements made by COPAS with manual estimations made of dithizone stained samples showed that the manual method consistently overestimated sample purity (Table 5).

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Islet Size Specific Analysis and Sorting

Studies of islet biology related specifically to intact islet size are limited due to the difficulty of obtaining a pure preparation of mono-sized islets in a rapid and practical manner. Semi-purified samples of human islets were stained with Newport Green and sorted based upon specific size ranges using the COPAS flow cytometer. As shown in Figure 2 (G-I), it was possible to sort islets of 50–100 μm, 150- 250 μm, and >250 μm size ranges at high purity with retention of intact morphology and dithizone staining.

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Newport Green Stained and COPAS Sorted Particles are Insulin Positive

To further assess the specificity of Newport Green staining and COPAS sorting for islets, we performed insulin immunohistochemistry on sorted particles. Human islets cultured for two days from the “bottom layer” of a continuous gradient purification of a pancreas digest were stained with Newport Green and sorted by fluorescence using the COPAS. The bottom layer fraction of this islet preparation was less than 25% pure and contained many trapped or mantled islets (data not shown). COPAS sorted green fluorescent particles were then either poststained with dithizone or fixed and embedded in paraffin for insulin immunohistochemistry (Fig. 3). The majority of sorted particles were identified as containing beta cells by dithizone or insulin staining.

FIGURE 3.

FIGURE 3.

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DISCUSSION

The difficulty of isolating pancreatic islets for the purposes of transplantation and research is compounded by the lack of an automated method of assessing the final islet yield and purity. At present, the most widely used method manually approximates islet size and purity by dithizone staining and optical microscopy. This method is prone to intra- and inter-reader variability, compromising the accuracy of the data within and across islet isolation facilities. The conflicting needs of an adequate sample size to accurately determine the IEQ count and a large islet mass at the time of transplantation, represents an unresolved clinical dilemma. In this study we evaluated large particle flow cytometry as a novel automated tool for the analysis of intact human islets and determination of accurate islet equivalent counts and purity. The primary objective is to present a new method that may reduce human-derived errors that compound the statistical limitations of small sample size.

Our initial experiments used uniform polystyrene microspheres as a means of testing the sensitivity and precision of the COPAS large particle flow cytometer when analyzing particles within the size range of intact islets. The COPAS has the capacity to analyze and sort particles based upon optical density, size, and three wavelengths of fluorescence. The time of flight (TOF) parameter measures the amount of time that a particle spends within the path of the excitation laser and is directly proportional to particle size. Particles are suspended within a laminar flow stream, which aligns them vertically by their longest dimension. The larger the particle size the greater the TOF value. Because of the parabolic nature of the sample streams flow rate within the sheath stream, particle size and TOF values do not increase linearly. Reflective of this fact was the observation that the most accurate line fit of particle size versus mean TOF value was achieved using a second order polynomial regression model. We used the polynomial regression line as a standard curve from which the diameters of particles of unknown size could be extrapolated. Microspheres comprising eight different diameters from 41.5 to 400 μm were measured with results within 5% of the manufacturer’s specifications. We also found that the fluidics and data acquisition components of the COPAS consistently produced sample recoveries greater than 94% for all microsphere sizes tested. These data show that the COPAS flow cytometer has the precision and accuracy required to detect, discriminate, and sample particles within the range of sizes typically observed for mammalian islets.

The manual method of islet equivalent (IEQ) calculation depends upon binning islets into 50 μm sizes (i.e., 50-100, 100-150, 150-200 μm, etc.) and then normalizing them to the volume of a 150-μm sphere. The inaccuracies of this method stem from its lack of objectivity due to errors made by the data recorder and that the total IEQ is not calculated based upon the exact volume of each individual islet, but rather an average of the number of islets grouped into each size range. We compared three different methods of calculating total IEQ yield of a simulated islet preparation containing a mixture of microspheres of varying sizes at known concentrations. The exact IEQ yield for the mock islet sample was calculated based upon the manufacturer’s size specifications and the known concentration of each microsphere size within the sample. Applying the standard method of IEQ yield determination by binning particles into 50-μm size ranges produced a 14.7% overestimate compared to the number calculated based upon the exact diameter of each individual particle in the sample. Samples of the mock islet preparation containing varying numbers of particles were analyzed by COPAS and the total IEQ yield calculated based upon the extrapolated diameter of each individual particle measured. When sample sizes of 300 particles or greater were measured the accuracy of the COPAS count was within 5% of the value calculated based upon the manufacturer’s specifications. The inaccuracy of the IEQ determination made by COPAS on a sample containing 100 particles likely reflects the difficulty of obtaining a truly representative sample of particles from a small volume (≤100 μl). These data demonstrate that the COPAS can accurately measure particles of varying sizes within a mixed sample, which allows for precise calculation of total particle volume and conversion to an IEQ count.

Utilization of the COPAS for islet assessments required an islet specific probe that would provide clear discrimination between islet and nonislet pancreatic tissue. Dithizone is a zinc specific dye that stains islet beta cells a dark red color and allows for their clear identification amongst contaminating nonislet tissue by optical microscopy. However, dithizone is nonfluorescent and its light absorbing properties make it suboptimal as an islet specific dye for flow cytometry. Newport Green has been shown by others to be an islet beta cell specific fluorescent probe that can be easily visualized by fluorescent microscopy and single-cell flow cytometry (7). We found that intact human islets could be easily and quickly loaded with Newport Green and their fluorescence emission discriminated from the autofluorescence of nonislet tissue by COPAS flow cytometry. The intensity of Newport Green staining was dependent upon islet size, islet viability, time in culture postisolation, and completeness of dye loading. Optimization of the conditions for dye loading and fluorescence detection allowed for clear discrimination between islet and nonislet tissue that was confirmed by dithizone staining and insulin immunohistochemical staining of sorted Newport Green positive particles. The majority of sorted particles that did not clearly stain with dithizone after sorting were observed to have heterogenous or light dithizone staining after washing in PBS (data not shown). Islets degranulate over time in culture leading to decreased intracellular zinc concentrations and diminished dithizone staining (11). This observation suggests that dithizone staining and optical microscopy might lack the sensitivity to detect islets containing beta cells with low intracellular zinc concentrations.

Having demonstrated the accuracy of measuring diverse particle sizes by COPAS flow cytometry and the ability to discriminate islets using Newport Green, we combined these methods into an automated method of determining an islet equivalent count on samples of six separate islet preparations. In five of six experiments the COPAS measured IEQ count was consistent with the count obtained by the standard manual method. In one experiment the COPAS IEQ count was twofold greater than the manual count. The counting disparity in this experiment was due to the presence of a significant percentage of islets embedded within acinar tissue. The TOF value measured for each Newport Green positive particle represents the length of the largest axis of the particle and not solely the islet. This observation presents a limitation of using Newport Green staining and COPAS flow cytometry to determine IEQ yields on islet preparations containing significant percentages of embedded islets. Further characterization of the correlation between fluorescent emission and islet size, may facilitate a method of compensating for the overestimation of the islet diameter in samples containing islets embedded within acinar tissue. Alternatively, the utilization of fluorescent probes specific for acinar tissue may allow for the accurate subtraction of the acinar tissue component that compromises the accuracy of the IEQ count.

Calculation of the ratio between IEQ and islet particles (IP) is one method of determining the average islet size in a preparation. An IEQ/IP ratio equal to 1 indicates an average islet diameter of 150 μm. From the data presented in Table 5 it is clear that the IEQ/IP ratios are slightly greater for the COPAS islet counts in comparison to the micrometer counts. In addition, although each Newport Green stained islet sample contained approximately 300 islet particles, the actual number analyzed and quantified averaged around 250. The discrepancies in both IEQ/IP ratios and islet particle counts reflect the exclusion of a portion of the islets ≤50 μm in diameter from the COPAS analysis. Although the total contribution of islets ≤50 μm to the total IEQ yield is not insignificant, in practice they are typically excluded by the manual counting method. Moreover, after culture the majority of islets ≤50 μm will contribute in high percentage to the single cell suspension mass, therefore they are irrelevant from the transplantation point of view. Despite these limitations, the automated COPAS flow cytometry method reduces the error in IEQ counting due to manual islet size estimation and volume calculation based upon size ranges rather than each individual particle’s diameter.

In addition to analyzing Newport Green stained islets for the purpose of IEQ determination we used the sorting capabilities of the COPAS to produce purified samples of islets of specific size ranges. We successfully sorted islets of 50–100 μm, 100–150 μm, and ≥250 μm with retention of morphology and capacity to stain with dithizone. This feature of the COPAS flow cytometer may allow further experimentation into the biology of islets based upon size.

Analysis of single cells by flow cytometry has become a common tool for both clinicians and basic science researchers. We provide the first data demonstrating that large particle flow cytometry is applicable to the study of intact isolated islets. As a quality control tool for clinicians, the COPAS can accurately determine IEQ counts and purities on preparations of nonembedded islets stained with Newport Green. This is an important feature because islet transplant success has been shown to partially depend upon islet graft mass (12). In addition, islet isolation center-to-center variance in islet yield quantification could be minimized using the automated COPAS IEQ determination method. As a tool for islet researchers the COPAS provides the ability to both enrich semipurified islet preparations from contaminating acinar tissue and select specific islet types based upon specific user defined criteria (e.g. size, plasma membrane, cytoplasmic, or nuclear markers). Expanding the capabilities of the COPAS will be accomplished by utilization of additional fluorescent probes of islet cell metabolism and function. Although our results show that islet morphology and staining capacity for dithizone are retained after COPAS analysis and sorting, additional studies are underway to determine the full functional capacity of these islets in vitro and in vivo.

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CONCLUSION

This is the first report showing the utilization of large particle flow cytometry for the analysis of intact live pancreatic islets. The COPAS flow cytometer was demonstrated to be highly accurate in the measurement of islet size allowing the determination of an islet equivalent count in an automated and computer-based method. Newport Green was shown to be an effective islet specific dye providing clear discrimination of islet from non-islet pancreatic tissue. Newport Green stained islets were purified into specific size groups using the sorting capabilities of the COPAS. The primary application of this method is currently the assessment of purified islet preparations. The accuracy of islet discrimination and size estimation did decrease when assessing islet preparations of low purity (data not shown), including preparations with mantled islets, in which there was an overestimation of islet size. The ability of the COPAS to purify islets from preparations with a high percentage of exocrine contamination is also limited at the present time. This report establishes the COPAS large particle flow cytometer as a tool that can be applied to both islet transplantation and research and could be used as a method for reliable quality control assessment.

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ACKNOWLEDGMENTS

The authors thank the University of Miami, Diabetes Research Institute, Miami, FL for assistance with islet isolation protocols.

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References

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Keywords:

Islet transplantation; Flow cytometry; Islet equivalent; Newport Green; Quality control

© 2005 Lippincott Williams & Wilkins, Inc.