Union, ME, United States
Union, ME, United States

Time filter

Source Type

Davis B.H.,Trillium Diagnostics LLC | Dasgupta A.,SRL Diagnostics Ltd | Kussick S.,PhenoPath | Han J.-Y.,Dong - A University | Estrellado A.,Biogen Idec
Cytometry Part B - Clinical Cytometry | Year: 2013

Flow cytometry and other technologies of cell-based fluorescence assays are as a matter of good laboratory practice required to validate all assays, which when in clinical practice may pass through regulatory review processes using criteria often defined with a soluble analyte in plasma or serum samples in mind. Recently the U.S. Food and Drug Administration (FDA) has entered into a public dialogue in the U.S. regarding their regulatory interest in laboratory developed tests (LDTs) or so-called "home brew" assays performed in clinical laboratories. The absence of well-defined guidelines for validation of cell-based assays using fluorescence detection has thus become a subject of concern for the International Council for Standardization of Haematology (ICSH) and International Clinical Cytometry Society (ICCS). Accordingly, a group of over 40 international experts in the areas of test development, test validation, and clinical practice of a variety of assay types using flow cytometry and/or morphologic image analysis were invited to develop a set of practical guidelines useful to in vitro diagnostic (IVD) innovators, clinical laboratories, regulatory scientists, and laboratory inspectors. The focus of the group was restricted to fluorescence reporter reagents, although some common principles are shared by immunohistochemistry or immunocytochemistry techniques and noted where appropriate. The work product of this two year effort is the content of this special issue of this journal, which is published as 5 separate articles, this being Validation of Cell-based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS - Part II - Preanalytical issues. © 2013 International Clinical Cytometry Society.


Liu Y.,Maine Institute for Human Genetics and Health | Hock J.M.,Maine Institute for Human Genetics and Health | Sullivan C.,Maine Institute for Human Genetics and Health | Fang G.,Maine Institute for Human Genetics and Health | And 4 more authors.
Journal of Cellular Biochemistry | Year: 2010

Arsenic trioxide (ATO) is a first-line anti-cancer agent for acute promyelocytic leukemia, and induces apoptosis in other solid cancer cell lines including breast cancer cells. However, as with arsenites found in drinking water and used as raw materials for wood preservatives, insecticides, and herbicides, low doses of ATO can induce carcinogenesis after long-term exposure. At 24 h after exposure, ATO (0.01-1 μM) significantly increased cell proliferation and promoted cell cycle progression from the G1 to S/G2 phases in the non-tumorigenic MCF10A breast epithelial cell line. The expression of 14 out of 96 cell-cycle-associated genes significantly increased, and seven of these genes including cell division cycle 6 (CDC6) and cyclin D1 (CCND1) were closely related to cell cycle progression from G1 to S phase. Low-dose ATO steadily increased gene transcript and protein levels of both CDC6 and cyclin D1 in a dose- and time-dependent manner. Low-dose ATO produced reactive oxygen species (ROS), and activated the p38 MAPK, Akt, and ERK1/2 pathways at different time points within 60 min. Small molecular inhibitors and siRNAs inhibiting the activation of p38 MAPK, Akt, and ERK1/2 decreased the ATO-increased expression of CDC6 protein. Inhibiting the activation of Akt and ERK1/2, but not p38 MAPK, decreased the ATO-induced expression of cyclin D1 protein. This study reports for the first time that p38 MAPK/Akt/ERK1/2 activation is required for the protein stabilization of CDC6 in addition to cyclin D1 in ATO-induced cell proliferation and cell cycle modulation from G1 to S phase. © 2010 Wiley-Liss, Inc.s.


Wong L.,Trillium Diagnostics LLC | Davis B.H.,Trillium Diagnostics LLC
Cytometry Part B - Clinical Cytometry | Year: 2013

Background: Assays of antigen expression on myeloid cells have an underlying premise that the assay integrates high purity gating of the leukocyte subpopulation in question. While CD45/side scatter (SSC) gating provides sufficient gating purity for qualitative assays of antigen expression; it is unsuitable for quantitative assays of antigen changes, especially monocytes. We have validated a monochromatic gating approach combining CD45 and CD64 labeled with the same fluorochrome that allows for high purity monocyte gating. Methods: Twenty-five blood samples were stained using three different antibody combinations (CD45 FITC + CD163 PE; CD45 FITC + CD64 PE; CD45 FITC + CD64 FITC). Data analysis focused on the percentage of "monocytes" defined by the various antibody and SSC gating combinations. Results: Percent monocyte recovered by monochromatic CD64 gating was not statistically different from two-color CD45 + CD64 or CD45 + CD163 gating. All three methods of immunologic monocyte identification yielded a 12.93%-15.15% reduction in the "monocyte" percentage compared to CD45/SSC gating. Conclusions: A monochromatic combination of CD45 and CD64 antibodies with scatter signals allows higher purity monocyte gating by flow cytometry (FC) compared to CD45/SSC gating. This approach allows for the development of a high resolution four-color assay, such as for detection of paroxysmal nocturnal hemoglobinuria, whereby a single four-color tube will allow simultaneous high purity monocyte (CD64+) and neutrophil (CD15+) analysis of both phosphatidylinositol (PI) linked protein expression and FLAER binding. © 2013 International Clinical Cytometry Society.


Wong L.,Trillium Diagnostics LLC | Hunsberger B.C.,Verity Software House | Bruce Bagwell C.,Verity Software House | Davis B.H.,Trillium Diagnostics LLC
International Journal of Laboratory Hematology | Year: 2013

Summary: Background: Flow cytometric methods (FCMs) are the contemporary standard for fetal red blood cell (RBC) quantitation and fetomaternal hemorrhage (FMH) detection. FCM provides greater sensitivity and repeatability relative to manual microscopic Kleihauer-Betke methods. FCM assays are not totally objective, employing subjective manual gating of fetal RBCs with measureable interobserver imprecision. We investigated Probability State Modeling to automate analysis of fetal RBCs using an assay for hemoglobin F (HbF)-containing RBCs. Methods: Two hundred human bloods were processed using the FMH QuikQuant™ assay (Trillium Diagnostics, Brewer, ME, USA). A Probability State Model (PSM) was designed to enumerate fetal RBCs by selecting the three RBCs subpopulation based on differences in intensity levels of several parameters. The GemStone™ program uses a PSM that requires no operator intervention. Routine manual analysis by experienced users was performed, along with replicate analyses for both methods. Results: The PSM by GemStone™ correlates strongly with the expert manual analysis, r2 = 0.9986. The mean absolute difference of the FMH results between GemStone™ and manual 'expert' analysis was 0.04% with no intermethod bias detected. Manual gating demonstrated coefficient of variations (CVs) of 10.6% for intra-analyst replicates and 22.6% for interanalyst imprecision. The interanalyst agreement in GemStone™ is a perfect correlation, r2 = 1.00, and no imprecision with a 0.00% CV. Conclusion: Automated PSM analysis of fetal RBCs strongly correlates with expert traditional manual analysis. PSM enumerates fetal RBCs accurately with significantly greater objectivity and lower imprecision than the traditional manual gating method. Thus, PSM provides a means to markedly improve interlaboratory variance with FMH assays based upon subjective gating strategies. © 2013 John Wiley & Sons Ltd.


Sutherland D.R.,University of Toronto | Acton E.,University of Toronto | Keeney M.,London Health Sciences Center | Davis B.H.,Trillium Diagnostics LLC | Illingworth A.,Dahl Chase Diagnostic Services
Cytometry Part B - Clinical Cytometry | Year: 2014

Background Recent Flow Cytometric guidelines to detect Paroxysmal Nocturnal Hemoglobinuria (PNH) in white blood cells recommend using FLAER-based assays to detect granulocytes and monocytes lacking expression of GPI-linked structures. However national proficiency testing results continue to suggest a need for improved testing algorithms, including the need to optimize diagnostic analytes in PNH. Methods CD157 is another GPI-linked structure expressed on both granulocytes and monocytes and here we assess its ability to replace CD24 and CD14 in predicate 4-color granulocyte and monocyte assays respectively. We also assess a single tube, 5-color combination of FLAER, CD157, CD64, CD15, and CD45 to simultaneously detect PNH clones in granulocyte and monocyte lineages. Results Delineation of PNH from normal phenotypes with 4- or 5-color CD157-based assays compared favorably with 4-color predicate methods and PNH clone size data were similar and highly correlated (R2 >0.99) with predicate values over a range (0.06%-99.8%) of samples. Both CD157-based assays exhibited similar high levels of sensitivity and low background levels in normal samples. Conclusions While CD157-based 4- and 5-color assays generated closely similar results to the predicate assays on a range of PNH and normal samples, the 5-color assay has significant advantages. Only a single 5-color WBC reagent cocktail is required to detect both PNH granulocytes and monocytes. Additionally, sample preparation and analysis time is reduced yielding significant efficiencies in technical resources and reagent costs. All 4- and 5-color reagent sets stained stabilized whole blood PNH preparations, used in external quality assurance programs. © 2013 International Clinical Cytometry Society © 2013 Clinical Cytometry Society.


Davis B.H.,Trillium Diagnostics LLC | Wood B.,University of Washington | Oldaker T.,Genoptix | Barnett D.,UK NEQAS
Cytometry Part B - Clinical Cytometry | Year: 2013

Flow cytometry and other technologies of cell-based fluorescence assays are as a matter of good laboratory practice required to validate all assays, which when in clinical practice may pass through regulatory review processes using criteria often defined with a soluble analyte in plasma or serum samples in mind. Recently the U.S. Food and Drug Administration (FDA) has entered into a public dialogue in the U.S. regarding their regulatory interest in laboratory developed tests (LDTs) or so-called "home brew" assays performed in clinical laboratories. The absence of well-defined guidelines for validation of cell-based assays using fluorescence detection has thus become a subject of concern for the International Council for Standardization of Haematology (ICSH) and International Clinical Cytometry Society (ICCS). Accordingly, a group of over 40 international experts in the areas of test development, test validation, and clinical practice of a variety of assay types using flow cytometry and/or morphologic image analysis were invited to develop a set of practical guidelines useful to in vitro diagnostic (IVD) innovators, clinical laboratories, regulatory scientists, and laboratory inspectors. The focus of the group was restricted to fluorescence reporter reagents, although some common principles are shared by immunohistochemistry or immunocytochemistry techniques and noted where appropriate. The work product of this two year effort is the content of this special issue of this journal, which is published as 5 separate articles, this being Validation of Cell-based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS - Part I - Rationale and aims. © 2013 International Clinical Cytometry Society.


Kottke-Marchant K.,Pathology and Laboratory Medicine Institute | Kottke-Marchant K.,Cleveland Clinic | Davis B.H.,Trillium Diagnostics LLC
Laboratory Hematology Practice | Year: 2012

Expertly edited and endorsed by the International Society for Laboratory Hematology, this is the newest international textbook on all aspects of laboratory hematology. Covering both traditional and cutting-edge hematology laboratory technology this book emphasizes international recommendations for testing practices. Illustrative case studies on how technology can be used in patient diagnosis are included. Laboratory Hematology Practice is an invaluable resource for all those working in the field. © 2012 Blackwell Publishing Ltd.


Briggs C.,University College London | Culp N.,Trillium Diagnostics LLC | Davis B.,Trillium Diagnostics LLC | d'Onofrio G.,Catholic University | And 2 more authors.
International Journal of Laboratory Hematology | Year: 2014

This revision is intended to update the 1994 ICSH guidelines. It is based on those guidelines but is updated to include new methods, such as digital image analysis for blood cells, a flow cytometric method intended to replace the reference manual 400 cell differential, and numerous new cell indices not identified morphologically are introduced. Haematology analysers are becoming increasingly complex and with technological advancements in instrumentation with more and more quantitative parameters are being reported in the complete blood count. It is imperative therefore that before an instrument is used for testing patient samples, it must undergo an evaluation by an organization or laboratory independent of the manufacturer. The evaluation should demonstrate the performance, advantages and limitations of instruments and methods. These evaluations may be performed by an accredited haematology laboratory where the results are published in a peer-reviewed journal and compared with the validations performed by the manufacturer. A less extensive validation/transference of the equipment or method should be performed by the local laboratory on instruments prior to reporting of results. © 2014 John Wiley & Sons Ltd.


A composition for evaluating a biological condition is disclosed. The composition has (a) a sample composition comprising at least one of: i.) a bodily specimen comprising a target moiety; ii.) a positive control moiety; and iii.) a negative control moiety; (b) an antibody composition comprising at least one of: i.) at least one target antibody; ii.) at least one positive control identifying antibody; and iii.) at least one negative control identifying antibody; and (c) at least one reference composition comprising at least one of: i.) a target signal reference composition; and ii.) a reference identifier composition.


The invention relates a method of quantifying CD64 and CD163 expression in leukocytes and, specifically to a kit for use with a flow cytometer including a suspension of quantitative fluorescent microbead standards, fluorescent labeled antibodies directed to CD64 and CD163, and analytical software. The software is used to take information on the microbead suspension and fluorescent labeled antibodies from a flow cytometer and analyse data, smooth curves, calculate new parameters, provide quality control measures and notify of expiration of the assay system.

Loading Trillium Diagnostics LLC collaborators
Loading Trillium Diagnostics LLC collaborators