Bruce H. Davis

Background Leuko64^TM (Trillium Diagnostics) is a flow cytometric assay that measures neutrophil CD64 expression and serves as an in vitro indicator of infection/sepsis or the presence of a systemic acute inflammatory response. Leuko64 assay currently utilizes QuantiCALC, a semiautomated software that employs cluster algorithms to define cell populations. The software reduces subjective gating decisions, resulting in interanalyst variability of TM (Verity Software House) and probability state modeling (PSM). Methods Four hundred and fifty-seven human blood samples were processed using the Leuko64 assay. Samples were analyzed on four different flow cytometer models: BD FACSCanto II, BD FACScan, BC Gallios/Navios, and BC FC500. A probability state model was designed to identify calibration beads and three leukocyte subpopulations based on differences in intensity levels of several parameters. PSM automatically calculates CD64 index values for each cell population using equations programmed into the model. GemStone software uses PSM that requires no operator intervention, thus totally automating data analysis and internal quality control flagging. Expert analysis with the predicate method (QuantiCALC) was performed. Interanalyst precision was evaluated for both methods of data analysis. Results PSM with GemStone correlates well with the expert manual analysis, r² = 0.99675 for the neutrophil CD64 index values with no intermethod bias detected. The average interanalyst imprecision for the QuantiCALC method was 1.06% (range 0.00–7.94%), which was reduced to 0.00% with the GemStone PSM. The operator-to-operator agreement in GemStone was a perfect correlation, r² = 1.000. Conclusion Automated quantification of CD64 index values produced results that strongly correlate with expert analysis using a standard gate-based data analysis method. PSM successfully evaluated flow cytometric data generated by multiple instruments across multiple lots of the Leuko64 kit in all 457 cases. The probability-based method provides greater objectivity, higher data analysis speed, and allows for greater precision for in vitro diagnostic flow cytometric assays. © 2015 International Clinical Cytometry Society

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

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

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.

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

Summary: The International Council for Standardization in Haematology (ICSH) in conjunction with Eurotrol, B.V. has released a new lot of the haemiglobincyanide or haemoglobin standard. This technical report describes the purpose, methodology in manufacturing, summary of value assignment data and availability of this standard material used for the standardization and calibration of whole blood haemoglobin measurements on most haemoglobinometers and automated blood cell counters throughout the world.

Translocation (8;21)/AML1-ETO is considered a favorable cytogenetic abnormality in acute myeloid leukemia (AML). However, associated KIT activating m

The clinical indications for diagnostic flow cytometry studies are an evolving consensus, as the knowledge of antigenic definition of hematolymphoid malignancies and the prognostic significance of antigen expression evolves. Additionally the standard of care is not routinely communicated to practicing clinicians and diagnostic services, especially as may relate to new technologies. Accordingly there is often uncertainty on the part of clinicians, payers of medical services, diagnostic physicians and scientists as to the appropriate use of diagnostic flow cytometry. In an attempt to communicate contemporary diagnostic utility of immunophenotypic flow cytometry in the diagnosis and follow‐up of patients with hematolymphoid malignancies, the Clinical Cytometry Society organized a two day meeting of international experts in this area to reach a consensus as to this diagnostic tool. This report summarizes the appropriate use of diagnostic flow cytometry as determined by unanimous approval of these experienced practitioners. © 2007 Clinical Cytometry Society

Sepsis, affecting millions of individuals annually with an associated high mortality rate, is among the top 10 causes of death. In addition, improvements in diagnostic tests for detecting and monitoring sepsis and infection have been limited in the last 25 years. Neutrophil CD64 expression has been proposed as an improved diagnostic test for the evaluation of infection and sepsis. To evaluate the diagnostic performance of a quantitative flow cytometric assay for leukocyte CD64 expression in comparison with the standard tests for infection/sepsis in an ambulatory care setting. Prospective analysis of 100 blood samples from patients from an emergency department setting in a 965-bed tertiary care suburban community hospital was performed for neutrophil CD64 expression, C-reactive protein, erythrocyte sedimentation rate, and complete blood count. The laboratory findings were compared with a clinical score for the likelihood of infection/sepsis, which was obtained by a blinded retrospective chart review. The diagnostic performance, as gauged by the clinical score, varied with neutrophil CD64 (sensitivity 87.9%, specificity 71.2%, efficiency 76.8%) and outperformed C-reactive protein (sensitivity 88.2%, specificity 59.4%, efficiency 69.4%), absolute neutrophil count (sensitivity 60.0%, specificity 50.8%, efficiency 53.8%), myeloid left shift (sensitivity 68.2%, specificity 76.3%, efficiency 73.3%), and sedimentation rate (sensitivity 50.0%, specificity 65.5%, efficiency 61.0%). Neutrophil CD64 expression quantitation provides improved diagnostic detection of infection/sepsis compared with the standard diagnostic tests used in current medical practice.

Context. —Sepsis, affecting millions of individuals annually with an associated high mortality rate, is among the top 10 causes of death. In addition, improvements in diagnostic tests for detecting and monitoring sepsis and infection have been limited in the last 25 years. Neutrophil CD64 expression has been proposed as an improved diagnostic test for the evaluation of infection and sepsis. Objective. —To evaluate the diagnostic performance of a quantitative flow cytometric assay for leukocyte CD64 expression in comparison with the standard tests for infection/sepsis in an ambulatory care setting. Design. —Prospective analysis of 100 blood samples from patients from an emergency department setting in a 965-bed tertiary care suburban community hospital was performed for neutrophil CD64 expression, C-reactive protein, erythrocyte sedimentation rate, and complete blood count. The laboratory findings were compared with a clinical score for the likelihood of infection/sepsis, which was obtained by a blinded retrospective chart review. Results. —The diagnostic performance, as gauged by the clinical score, varied with neutrophil CD64 (sensitivity 87.9%, specificity 71.2%, efficiency 76.8%) and outperformed C-reactive protein (sensitivity 88.2%, specificity 59.4%, efficiency 69.4%), absolute neutrophil count (sensitivity 60.0%, specificity 50.8%, efficiency 53.8%), myeloid left shift (sensitivity 68.2%, specificity 76.3%, efficiency 73.3%), and sedimentation rate (sensitivity 50.0%, specificity 65.5%, efficiency 61.0%). Conclusion. —Neutrophil CD64 expression quantitation provides improved diagnostic detection of infection/sepsis compared with the standard diagnostic tests used in current medical practice.