Standardization of immunophenotyping requires careful attention to reagents, sample handling, instrument setup, and data analysis, and is essential for successful cross-study and cross-center comparison of data. Experts developed five standardized, eight-color panels for identification of major immune cell subsets in peripheral blood. These were produced as pre-configured, lyophilized, reagents in 96-well plates. We present the results of a coordinated analysis of samples across nine laboratories using these panels with standardized operating procedures (SOPs). Manual gating was performed by each site and by a central site. Automated gating algorithms were developed and tested by the FlowCAP consortium. Centralized manual gating can reduce cross-center variability, and we sought to determine whether automated methods could streamline and standardize the analysis. Within-site variability was low in all experiments, but cross-site variability was lower when central analysis was performed in comparison with site-specific analysis. It was also lower for clearly defined cell subsets than those based on dim markers and for rare populations. Automated gating was able to match the performance of central manual analysis for all tested panels, exhibiting little to no bias and comparable variability. Standardized staining, data collection, and automated gating can increase power, reduce variability, and streamline analysis for immunophenotyping.

In the above article, we did not include a GEO accession number for the gene expression data from our study. The expression data can be accessed under the accession number GSE47353 at http://www.ncbi.nlm.nih.gov/geo/. In addition, all data used in our analyses can be obtained at http://chi.nhlbi.nih.gov.

Storing platelets in an additive solution containing magnesium and potassium improves the functionality of the platelets, as measured by in vitro testing, and may allow a reduction of the amount of plasma required to be carried over to the final unit.

Introduction Cytokines are humoral regulatory molecules that act together in immunologic pathways. Monitoring cytokines and their variations within physiologic ranges is critical for biomarker discovery. Therefore, we evaluated the performance characteristics of 72 analytes measured by multiplex cytokine immunoassay, with an emphasis on the differences of analytes measured in serum compared to plasma, and, in plasma, on the impact of anticoagulants on the cytokine measurement. Methods We used fluorescent bead-based (Luminex) immunoassay kits to simultaneously measure 72 analytes. We tested serum and plasma samples from 11 matched donors. Plasma samples were anti-coagulated with sodium heparin, sodium citrate dextrose and ethylene diamine tetra-acetic acid (EDTA), respectively. Results Of the 72 cytokines, 12 were undetectable in all types of specimen samples. Nineteen analytes, including PDGF-bb, IL-4, IL-8, IL-9, FGF-b, PAI-1, CXCL-5, CCL-5, CD40L, EGF, VEGF, IL-2ra, IL-3, SDF-1a, PCT, MCP-3, GIP, IL-16 and fibrinogen, showed significant differences between measurements in serum and all types of plasma, regardless of anticoagulant. Among plasma samples, 10 analytes (eotaxin, SCGF-b, MCP-1, SCF, MIP-1b, VEGF, RANTES, PDGF-b, PAI-1 and ITAC) showed significantly higher concentrations in heparinized plasma compared to citrated and EDTA plasma. IP-10, and CTAK were the only 2 cytokines that presented different concentrations in citrate and EDTA plasma. Conclusions With their small volume, low cost per test, and multiplex capacity, Luminex-based cytokine assays have enormous potential utility for screening in epidemiologic studies. In our study, we showed that many cytokines’ concentrations differed between serum and plasma samples, and that different anticoagulants used in preparation of plasma samples also affected the measurement of some cytokines. There was no optimal sample preparation that was clearly superior for the measurement of all analytes measured. Ultimately, the utility of cytokine measurement, as biomarker or to monitor the immune system, will depend on attention to detail in the collection and processing of samples in addition to assay precision.

BackgroundCytokines are humoral molecules that elicit regulatory function in immunologic pathways. The level and type of cytokine production has become critical in distinguishing physiologic from pathologic immune conditions. Cytokine profiling has become an important biomarker discovery tool in monitoring of the immune system. However, the variations in cytokine levels in individual subjects over time in healthy individuals have not been extensively studied. In this study, we use multiplex bead arrays to evaluate 27 analytes in paired serum samples taken seven days apart from 144 healthy individuals in order to assess variations over a short time period.MethodsFluorescent bead-based immunoassay (Luminex) was used to measure 27 analytes in serum samples. Measurements were performed on matched samples from 144 healthy donors. To assess inter-plate variability, one arbitrarily selected serum sample was analyzed on each of the first ten plates as bridge sample. ResultsUsing the bridge sample, we showed minimal inter-plate variations in the measurement of most analytes. In measurement of cytokines from the 144 patients at two time points, we found that three cytokines (IL-2, IL-15 and GM-CSF) were undetectable and five analytes (RANTES, MCP-1, VEGF, MIP-1β and PDGF-BB) showed significant difference in concentrations at Day 0 compared to Day 7. ConclusionsThe current study demonstrated higher variations in cytokine levels among individuals than were observed for samples obtained one week apart from identical donors. These data suggest that a serum sample from each subject for use as a baseline measurement is a better control for clinical trials rather than sera from a paired cohort.

Flow cytometric (FCM) reticulocyte analysis is more accurate, sensitive, and reproducible relative to previously employed manual microscopic methods in clinical laboratory hematology. FCM reticulocyte analysis using RNA binding fluorochromes additionally allows for the quantification of fluorescence intensity or population distribution of the reticulocyte RNA content. Viewed from the perspective of erythroid maturation, quantification of the fluorescence intensity distribution provides a reticulocyte maturation index (RMI). We performed a systematic study of 18 different methods to express thiazole orange stained reticulocyte fluorescence intensity, compared to standard mean fluorescence intensity quantification, using 185 anemic and non-anemic human blood samples, The method best correlating with the mean fluorescence intensity RMI on 2 different FCM instruments (R2 = 0.93 and 0.86) was a ratio of the highly fluorescent reticulocytes, defined using a normal adult population, and the total number of reticulocytes (HFR%). In contrast to mean fluorescence intensity measurements, a HFR% RMI parameter can provide similar units of expression (0.01-1.00) with good correlation between different FCM instruments (R2 = 0.76). We conclude the HFR% method of RMI expression provides a superior means of interlaboratory standardization and clinical comprehension of this useful diagnostic parameter in clinical laboratory hematology. 0 1993 Wiley-Liss, Inc.Keyterms: Red blood cell, anemia, erythrocyte, fluorescent dyes, blood, eryth-ropoiesis Clinical flow cytometric reticulocyte (FCM retic) analysis is now widely practiced in clinical laboratories in the United States, Europe, and Japan. In terms of numbers of patients, FCM retic analysis likely represents the most common clinical application of fluorescence-based flow cytometry. Interlaboratory standardization of this assay is currently of great concern in laboratory hematology. A subcommittee of the National Committee for Clinical Laboratory Standards is developing guidelines for this form of clinical testing. The American Medical Association CPT coding system now recognizes reticulocyte analysis by flow cytometry as a diagnostic medical test, distinct from manual microscopic counting methodology.FCM retic can be performed with a number of methods [10,25], utilizing fluorescent dyes such as thiazole orange [4,8,9,12,17], thioflavin T [5,19,21], ethidium bromide [51, pyronin Y [24,271, and acridine orange [10,19,22,23,33-351. In addition, TOA Medical Electronics (Kobe, Japan) markets a dedicated, semiautomated reticulocyte counter using flow cytometric technology and the fluorescent dye auramine 0 on the Sysmex R-1000 instrument [16,28,291. All of these methods of reticulocyte enumeration give excellent correlation (R2 range: 0.80-0.95) with one another [lo].Laboratory methods of quality control of clinical FCM retic analysis have been proposed using refrigerated rabbit blood [5,61, normal human samples [9,10,3 11, and commercial reticulocyte control reagents (Streck Labo...

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N-formylated chemotactic peptide stirnulation of human neutrophils initiates a number of cellular processes, such as lysosomal enzyme release and superoxide anion production, that are indicative of the events of neutrophil activation during the acute inflammatory response in disease. This study characterizes a newly recognized neut,rophil activation event, N-formylated chemotactic peptide-stimulated fluid pinocytosis in human neutrophils, using a novel flow cytornetric assay for this activity. Fluid pinocytosis was found to be inhibited by acidic pH and low temperature but could be enhanced by cytochalasin R treatment or surface adherence by neutrophils. The activity measured by this new assay of fluid pinocytosis appears to be separate and distinct from lysosomal enzyme release and receptor-mediated adsorptive endocytosis in neutrophils. The physiologic significance of N-formylated chemotactic peptide-stimulated f h i d pinocytosis is not known, but a possible relationship to neutrophi1 locomotion is discussed.Key terms: Neutrophils, chernotactic factors, neutrophil activation, endocytosis, chemotaxis Neutrophils or polymorphonuclear leukocytes (PMN) human neutrophils using flow cytometric analysis of the serve as a major effector cell in host defense and acute process. inflammation. Many of the cellular, physiologic, and Stimulation of pinocytotic activity, induced by ligandbiochemical responses or activation events of these cells membrane receptor interactions, occurs in a number of have been elucidated in an in vitro model system using mammalian cells, such as in fibroblasts by epidermal N-formyl chemotactic peptides as the stimulus (3,5,27-growth factor (24) and low-density lipoprotein (151, neu-30). Therefore, in vitro studies using PMN and N-for-rons by nerve growth factor (€0, and hepatocytes by mylated peptides, such as N-formyl-methionyl-leucyl-asialoglycoproteins (31). Often a clear distinction bephenylalanine (fMLP), continue to contribute signifi-tween the two types of cellular endocytosis, fluid pinocantly to the basic understanding of cellular processes cytosis, and adsorptive endocytosis is not made in studies involved in inflammatory disease.of ligand-stimulated endocytic activity. In fact, the focus Stimulation of fluid pinocytosis (FP) by the chemotac-of all but one (10) of the studies of pinocytosis in neutrotic formyl peptides in polymorphonuclear leukocytes is phils, published to date, has been the delineation of a newly recognized phenomenon related to neutrophil adsorptive endocytosis in rabbit (9,291 and human (13, activation (9,10,29). It is clear from published studies 16,17) PMN. Since previous work (9,101 has demonthat stimulated pinocytosis in rabbit and human PMN occurs in a concentration range of N-formyl peptide similar to that which initiates other events of neutrophil activation, such as chemotaxis (301, lysosomal enzyme release (2), oxidative metabolism burst (5,18), and membrane Potential changes (25). However, the relationship of FP to other known events of neutrophil activ...