Functional assessment of automatically sorted pancreatic islets using large particle flow cytometry

The size composition of human islet preparations has been attributed to functional potency, islet survival and transplantation outcomes. In the early post-transplantation phase islets are supplied with oxygen by diffusion only and are at risk of critical hypoxia. The high rate of early islet graft dysfunction is in part attributed to this condition. It has been presumed that islets with smaller diameter, and therefore smaller diffusion distance, are superior to large islets regarding early survival rate and graft function. In this study we aimed to evaluate Complex Object Parametric Analysis and Sorting (COPAS) as a device for automated sorting of human islets. The use of COPAS was validated for accuracy and sensitivity using polystyrene beads of known diameters. Based on time of flight relative to particle isolated islets were then automatically sorted and analyzed for viability and function using handpicked islets as control. Our results suggest that COPAS enables the automated and accurate sorting of islets with no negative impact on their integrity and viability. Thus, COPAS is an adequate tool for size-specific analysis of pancreatic islets and may be considered as part of a platform for automated high-throughput screening of pancreatic islets.     

Functional assessment and specific depletion of alloreactive human T cells using flow cytometry

Human T-cell alloreactivity plays an important role in many disease processes, including the rejection of solid organ grafts and graft-versus-host disease (GVHD) following allogeneic stem cell transplantation. To develop a better understanding of the T cells involved in alloreactivity in humans, we developed a cytokine flow cytometry (CFC) assay that enabled us to characterize the phenotypic and functional characteristic of T cells responding to allogeneic stimuli. Using this approach, we determined that most T-cell alloreactivity resided within the CD4(+) T-cell subset, as assessed by activation marker expression and the production of effector cytokines (eg, tumor necrosis factor alpha [TNF]alpha) implicated in human GVHD. Following prolonged stimulation in vitro using either allogeneic stimulator cells or viral antigens, we found that coexpression of activation markers within the CD4(+) T-cell subset occurred exclusively within a subpopulation of T cells that significantly increased their surface expression of CD4. We then developed a simple sorting strategy that exploited these phenotypic characteristics to specifically deplete alloreactive T cells while retaining broad specificity for other stimuli, including viral antigens and third-party alloantigens. This approach also was applied to specifically enrich or deplete human virus-specific T cells.     

Functional homogeneity of pancreatic islets of aging rats

Islets from different regions of pancreases of aging rats were compared for size and variations in response to glucose stimulation. The results show that pancreatic islets from the ventral-duodenal and splenic regions of 12-mo-old retired breeder Spraque-Dawley rats are comparable in all respects measured: thus, pancreatic regional differences cannot explain the age-associated reduction in beta cell secretory response noted in previous studies.     

Reticulocyte analysis using flow cytometry

Automation of the reticulocyte count by means of flow cytometry has considerably improved the quality of this investigation. This article deals firstly with the reasons for the poor performance of the microscopic technique and with the physiological principles underlying identification and classification of reticulocytes using RNA labeling. It then outlines the automated methods currently on the market, which can be classified in three categories: a) general-purpose cytofluorometers, which in clinical laboratories usually deal with lymphocyte immunophenotyping; b) the only commercially available cytofluorometer dedicated to the reticulocyte count; this automat has the advantage of requiring no human intervention as it merely needs to be fed with samples; c) hematology analyzers with specific modules for automatic counting of reticulocytes previously incubated with a non-fluorescent dye. Of the various fluorescent markers available, thiazole orange, DEQTC iodide and auramine are most often used for this basic hematology test. The quality of the count, the availability of new reticulocyte indices (maturation index, percentage of young reticulocytes) and the rapidity of the count give this test renewed value in the practical approach to the diagnosis of anemia, and also open new perspectives in the surveillance of aplastic anemia after chemotherapy or bone marrow grafting.     

Reticulocyte analysis using flow cytometry

Automation of the reticulocyte count by means of flow cytometry has considerably improved the quality of this investigation. This article deals firstly with the reasons for the poor performance of the microscopic technique and with the physiological principles underlying identification and classification of reticulocytes using RNA labeling. It then outlines the automated methods currently on the market, which can be classified in three categories: a) “general-purpose” cytofluorometers, which in clinical laboratories usually deal with lymphocyte immunophenotyping; b) the only commercially available cytofluorometer dedicated to the reticulocyte count; this automat has the advantage of requiring no human intervention as it merely needs to be fed with samples; c) hematology analyzers with specific modules for automatic counting of reticulocytes previously incubated with a non-fluorescent dye. Of the various fluorescent markers available, thiazole orange, DEQTC iodide and auramine are most often used for this basic hematology test. The quality of the count, the availability of new reticulocyte indices (maturation index, percentage of young reticulocytes) and the rapidity of the count give this test renewed value in the practical approach to the diagnosis of anemia, and also open new perspectives in the surveillance of aplastic anemia after chemotherapy or bone marrow grafting.     

Functional assessment of anomalous right coronary artery using fractional flow reserve

Anomalous origin of a coronary artery is a recognized cause of sudden cardiac death (SCD). To date, there is no standard test to predict which patients are at increased risk for SCD. Fractional flow reserve (FFR) is an invasive technique used to qualify focal obstructive coronary lesions. We present a case where FFR was used to guide therapy in a young patient with anomalous right coronary artery (ARCA) when standard noninvasive testing showed ischemic discrepancy. © 2016 Wiley Periodicals, Inc.     

Immunogold probing of platelet factor 4 in different ploidy classes of rat megakaryocytes sorted by flow cytometry

Different ploidy classes of rat megakaryocytes were sorted by flow cytometry from highly purified perfusion-fixed megakaryocyte cell suspensions prepared by sequential centrifugal elutriation and Percoll gradient centrifugation. Sorted cell populations were studied for the localization of platelet factor 4 (PF-4) probed with the monoclonal antibody 2E7 in order to clarify the relevance of PF-4 localization to the cytoplasmic and nuclear development of megakaryocytes. The relative numbers of labeled alpha granules and labeled alpha granule-related small vesicular structures (AGR-SVS) were quantitated using the gold-labeled antibody detection method and correlated with DNA content and cytoplasmic maturation in individual megakaryocytes. We determined that the stage of cytoplasmic maturation exerted a significant effect on the proportion of labeled alpha granules and labeled AGR-SVS. A significant interaction effect of stage and ploidy class resulted in the stage effect on proportion of labeled alpha granules being significant only in two of the three ploidy classes. The least mature cells present within each ploidy group exhibited PF-4 labeling mostly in SVS that were not related to alpha granules. During subsequent cytoplasmic maturation, more of the labeled SVS were seen related to alpha granules, with more of the mature alpha granules themselves becoming labeled. Polyploidization also affected the proportion of labeled AGR-SVS. Our data suggest that SVS play a role in the intramegakaryocytic transport of PF-4 into alpha granules. These data provide evidence of the complexity of megakaryocytic differentiation involving both cytoplasmic maturation and nuclear endoreduplication as reflected in PF-4 expression.     

Rapid assessment of hyperdiploidy in plasma cell disorders using a novel multi-parametric flow cytometry method

Trisomies of odd numbered chromosomes are seen in nearly half of patients with multiple myeloma (MM) and typically correlate with a hyperdiploid state and better overall survival (OS). We compared DNA ploidy of monoclonal plasma cells (as a surrogate for the presence of trisomies) assessed simultaneously by PCPRO (plasma cell proliferative index), a novel method that estimates DNA index by multi-parametric flow cytometry to fluorescence in situ hybridization (FISH) in 1703 patients with plasma cell disorders. The distribution of ploidy was hyperdiploid: 759 (45%), diploid 765 (45%), hypodiploid: 71 (4%), tetraploid/near-tetraploid: 108 (6%). FISH identified trisomies in 82% (621/756) of patients with hyperdiploidy by PCPRO and no trisomy by FISH was observed in 88% (730/834) of patients without hyperdiploidy. 95% (795/834) of patients without hyperdiploidy on PCPRO had one or less trisomy by FISH. Sensitivity and specificity of PCPRO for detecting hyperdiploidy was 86% (621/725) and 84% (730/865), respectively. Sensitivity increased to 94% (579/618) for patients with more than one trisomy. Newly diagnosed MM patients with hyperdiploidy on PCPRO (147/275) had better OS compared to nonhyperdiploid patients (median not reached vs 59 months, P = 0.008) and better progression free survival (median: 33 vs 23 months, P = 0.03). Within the hyperdiploidy group, patients with high-hyperdiploidy (DNA index: 1.19-1.50) versus those with low-hyperdiploidy (DNA index: 1.05-1.18) had superior OS (3 year OS of 88% vs 68% P = 0.03). Ploidy assessment by flow cytometry can provide rapid, valuable prognostic information and also reduces the number of copy number FISH probes required and hence the cost of FISH.     

Endocrine pancreas engineered using porcine islets and partial pancreatic scaffolds

ObjectivesBecause therapeutic options for severe diabetes are currently limited, there is a continuing need for new therapeutic strategies, especially in the field of regenerative medicine. Collaborative efforts across the fields of tissue engineering technology and islet biology may be able to create functionally engineered islets capable of restoring endocrine function in patients with insulin-dependent diabetes.MethodsThis engineered scaffold was seeded with isolated primary porcine islets via the pancreatic duct using a multi-step infusion technique. Endocrine function of perfusion-cultured islets in the native scaffold was analyzed by immunohistochemical staining of insulin and glucagon as well as by the insulin stimulation test.ResultsThe pancreas in this large animal could be uniformly decellularized by perfusion with trypsin and TritonX-100 via the pancreatic duct, as shown by positive staining of extracellular matrix (ECM) components. These scaffolds derived from porcine pancreas were able to maintain the cellular integrity of islets that had repopulated the parenchymal space, which is fundamental for the restoration of endocrine function. Insulin release up to four days after islet infusion was maintained.ConclusionsThis scaffold from a large animal maintained islet survival and function in the short-term, retaining the cells as a solid organ in the parenchymal space after infusion through the pancreatic duct. These results suggest that this scaffold is suitable for further fabrication of fully functional bioengineered endocrine pancreases when implanted in vivo. Therefore, it may represent a key improvement in the field of beta-cell replacement therapy. Nonetheless, the facilitation of longer-term islet survival and studies of implantation in vivo is required for successful clinical translation.     

Functional characterization of alpha-adrenoceptors on pancreatic islets of fa/fa Zucker rats.

Recently, a defect in pertussis toxin-independent actions of epinephrine on pancreatic B-cells of fa/fa Zucker rats was reported (Cawthorn and Chan (1991) Mol. Cell. Endocrinol. 75, 197-204). We now report studies of islet alpha 2-adrenoceptor function of fa/fa rats. Insulin and cAMP production by islets of obese rats were both inhibited by the alpha 2-adrenoceptor agonist clonidine. Calculated pD2 values for clonidine were 9.57 +/- 0.59 and 9.43 +/- 0.33 for lean and fa/fa rat islets, respectively. Yohimbine reversed clonidine effects equipotently in lean and obese rat islets (pA2 values of 7.48 +/- 0.57 vs 7.43 +/- 0.58). Unexpectedly, the alpha 1-antagonist prazosin stimulated insulin secretion from islets of obese but not lean rats. Functional characteristics of the alpha-adrenoceptors on fa/fa islets are thus similar to those recently designated alpha 2B. Altered expression of alpha-adrenoceptors on pancreatic islets of fa/fa rats may contribute to changes in the pertussis toxin-independent pathway of epinephrine action previously observed.     

Regenerative medicine of pancreatic islets

The pancreas became one of the first objects of regenerative medicine,since other possibilities of dealing with the pancreatic endocrine insufficiency were clearly exhausted.The number of people living with diabetes mellitus is currently approaching half a billion,hence the crucial relevance of new methods to stimulate regeneration of the insulin-secreting β-cells of the islets of Langerhans.Natural restrictions on the islet regeneration are very tight;nevertheless,the islets are capable of physiological regeneration via β-cell self-replication,direct differentiation of multipotent progenitor cells and spontaneous α-to or δ-to β-cell conversion(trans-differentiation).The existing preclinical models of β-cell dysfunction or ablation(induced surgically,chemically or genetically) have significantly expanded our understanding of reparative regeneration of the islets and possible ways of its stimulation The ultimate goal,sufficient level of functional activity of β-cells or their substitutes can be achieved by two prospective broad strategies β-cell replacement and β-cell regeneration.The "regeneration" strategy aims to maintain a preserved population of β-cells through in situ exposure to biologically active substances that improve β-cell survival,replication and insulin secretion,or to evoke the intrinsic adaptive mechanisms triggering the spontaneous non-β-to β-cell conversion.The "replacement" strategy implies transplantation of β-cells(as non-disintegrated pancreatic material or isolated donor islets) or β-like cells obtained ex vivo from progenitors or mature somatic cells(for example,hepatocytes or a-cells) under the action of small-molecule inducers or by genetic modification.We believe that the huge volume of experimental and clinical studies will finally allow a safe and effective solution to a seemingly simple goal-restoration of the functionally activeβ-cells, the innermost hope of millions of people globally.     

Metabolism of cold-stored pancreatic islets

Summary A previous study showed that the ability of glucose to stimulate insulin release was retained in islets stored at 8 °C for one week provided that glucose was present in a high concentration in the storage medium. The metabolic properties of islets stored in the cold have now been further explored in an attempt to clarify the protective effect of glucose. During storage in the cold the islet formation of ³H₂O from (5-³H) glucose and oxygen consumption were only a few per cent of that of fresh islets whereas the uptake of ⁸⁶Rb⁺ was 20–48%. Rewarming the cold-stored islets to 37 °C after one week of cold-storage restored the ⁸⁶Rb⁺ uptake, the formation of ³H₂O and ¹⁴CO₂ from labelled glucose and oxygen consumption to 75, 80, 60 and 40% respectively of fresh islet levels. The results emphasize the usefulness of cold-storage for preservation of functionally intact isolated islets.On leave from the Metabolic Research Unit, 1143 HSW, University of California, San Francisco, CA 94143, USA     

Functional impact of attachment and purification in the short term culture of human pancreatic islets

We have evaluated the effects on islet function of several manipulations of the substrate and tissue culture conditions in the short term culture of human islets. Specifically, we have studied the influence of several matrices, additions to the medium, and the use of basic fibroblast growth factor (FGF)-saporin mitotoxins to eliminate fibroblastoid cells from the cultures. The human islets were obtained from the Human Islet Transplant Center at Washington University Medical Center (St. Louis, MO). Substrates used to facilitate islet attachment were poly-L-lysine, gelatin, Matrigel, collagen, and bovine corneal endothelial cell matrix. RPMI-1640 medium was supplemented with either 22.2 mM glucose or 10 micrograms/mL human insulin. FGF-saporin mitotoxin was used at a concentration of 10 nM. The greatest improvement in islet cell function in either static or stimulated situations was obtained when we used bovine corneal endothelial cell matrix as the matrix, supplemented the medium with a high concentration of glucose or insulin, and eliminated fibroblast-like cells by exposing the cultures to basic FGF-saporin mitotoxin. The conditions described in this report could greatly improve the culture of human islets for use in clinical and laboratory research.     

Stimulation by glucose of the blood flow to the pancreatic islets of the rat

Diabetologia - Blood flow to the pancreatic islets of the rat was estimated with the microsphere technique. Experiments with microspheres of different sizes (diameter 10, 15 or 50 μm) showed...     

Investigation of platelet function and platelet disorders using flow cytometry

Patients with thrombocytopenia or platelet disorders are at risk of severe bleeding. We report the development and validation of flow cytometry assays to diagnose platelet disorders and to assess platelet function independently of platelet count. The assays were developed to measure glycoprotein levels (panel 1) and platelet function (panel 2) in sodium citrated blood. Twenty healthy volunteers and five patients diagnosed with different platelet disorders were included. Glycoprotein expression levels of the receptors Ia, Ib, IIb, IIIa and IX were measured and normalised with forward scatter (FS) as a measurement of platelet size. Platelet function was assessed by CD63, P-selectin and bound fibrinogen in response to arachidonic acid, adenosine diphosphate (ADP), collagen-related peptide, ristocetin and thrombin receptor-activation peptide-6. All patients except one with suspected δ-granule defect showed aberrant levels of glycoproteins in panel 1. Glanzmann's thrombasthenia and genetically verified Bernard–Soulier syndrome could be diagnosed using panel 1. All patients showed reduced platelet function according to at least one agonist. Using panel 2 it was possible to diagnose Bernard–Soulier syndrome, δ-granule defect and GPVI disorder. By combining the two assays, we were able to diagnose different platelet disorders and investigate platelet function independent of platelet count.     

Rapid quantitation of mixed red cell populations using flow cytometry

Methods are described for the identification and quantitation of mixed red cell populations using flow cytometry. Antibodies specific for a wide range of blood group antigens have been used and examples are given in which these analyses have proved to be of clinical use. These examples include monitoring of erythropoiesis following engraftment in allogeneic bone marrow transplant recipients and the detection of chimaeric states months or years after transplantation. The techniques involved are fast, simple and inexpensive.