Cell surface receptor-antibody association constants and enumeration of receptor sites for monoclonal antibodies

BACKGROUND: Fluorescent markers (labeled antibodies) and flow cytometry are used to enumerate the average number of receptors (antigens) on formed bodies (cells) in whole blood by using a new method that avoids the extra steps of separating bound from unbound fluorescent markers or the use of external standards. METHODS: Mean channel fluorescence intensities of equilibrated marker-cell suspension mixtures, total concentrations of marker, and targeted cell counts obtained by standard cytometry procedures are used to complete the analyses for receptors per cell. Also, flow cytometric assays using competitive binding between fluorescent marker (CD4-RD1, CD8-FITC, CD3-FITC, CD3-RD1) and unlabeled antibody (CD4, CD8, CD3, CD3-dextran) for receptors on white blood cells in whole blood are described for determination of relative and specific binding constants of unlabeled/labeled antibody for targeted receptors. RESULTS: Ranges that were obtained for receptors per cell (lymphocytes) in normal blood donors were as follows: CD4, 4.9 x 10(4)-1.5 x 10(5); CD8, 5.0 x 10(5)-2.1 x 10(6); CD3, 6.6-7.8 x 10(5). Binding constants were highest for unlabeled CD4 antibody, 2. 7 x 10(10)-2.1 x 10(12) M(-1), and then unlabeled CD3 antibody, 1.1 x 10(10)-1.9 x 10(11) M(-1). FITC- and RD1-labeled antibodies typically had binding constants that were 10-to 100-fold lower than the native antibodies. CONCLUSIONS: Values of receptors per cell and binding constants obtained by the new method from flow cytometric analyses of mixtures of whole blood with FITC- or RD1-labeled CD4, CD8, and CD3 antibodies compare well with literature values determined by other methods.     

Human CD4+ lymphocytes for antigen quantification: characterization using conventional flow cytometry and mass cytometry

To transform the linear fluorescence intensity scale obtained with fluorescent microspheres to an antibody bound per cell (ABC) scale, a biological cell reference material is needed. Optimally, this material should have a reproducible and tight ABC value for the expression of a known clinical reference biomarker. In this study, we characterized commercially available cryopreserved peripheral blood mononuclear cells (PBMCs) and two lyophilized PBMC preparations, Cyto-Trol and PBMC-National Institute for Biological Standard and Control (NIBSC) relative to freshly prepared PBMC and whole blood samples. It was found that the ABC values for CD4 expression on cryopreserved PBMC were consistent with those of freshly obtained PBMC and whole blood samples. By comparison, the ABC value for CD4 expression on Cyto-Trol is lower and the value on PBMC-NIBSC is much lower than those of freshly prepared cell samples using both conventional flow cytometry and CyTOF? mass cytometry. By performing simultaneous surface and intracellular staining measurements on these two cell samples, we found that both cell membranes are mostly intact. Moreover, CD4(+) cell diameters from both lyophilized cell preparations are smaller than those of PBMC and whole blood. This could result in steric interference in antibody binding to the lyophilized cells. Further investigation of the fixation effect on the detected CD4 expression suggests that the very low ABC value obtained for CD4(+) cells from lyophilized PBMC-NIBSC is largely due to paraformaldehyde fixation; this significantly decreases available antibody binding sites. This study provides confirmation that the results obtained from the newly developed mass cytometry are directly comparable to the results from conventional flow cytometry when both methods are standardized using the same ABC approach.     

Competitive antibody binding to soluble CD16B antigen and CD16B antigen on neutrophils in whole blood by flow cytometry

BACKGROUND: Shed receptors from the surface of white blood cells in whole blood have been quantitated using the long and tedious enzyme-linked immunosorbent assay (ELISA) method. A simple rapid flow cytometric method of analysis for shed antigen in the presence of cell-bound antigen can be advantageous. METHODS: Magnetic bead depletion of neutrophils in whole blood with CD16b antibody-conjugated beads as measured by flow cytometric analysis of the remaining cell suspension was inhibited by the presence of soluble CD16b antigen in the blood plasma of normal donors. We describe a competitive binding assay between labeled and unlabeled CD16b antibody for receptors shed from the surface of formed bodies (cells) into solution. Also presented is a new method of obtaining the amount of soluble antigen in a sample. We determine the total unlabeled and labeled ligand concentration at which the fluorescence intensity of the labeled ligand matches the fluorescence intensity in a control run with only the labeled ligand. RESULTS: Normal blood donors showed serum concentration levels of shed CD16b antigen in the range of 1-50 nM as determined by a flow cytometric competitive binding assay. These figures compared favorably with parallel determinations using magnetic bead depletion of targeted neutrophils for washed and unwashed whole blood samples to evaluate the concentration of shed CD16b antigen. CONCLUSIONS: The competitive antibody binding assay for shed and cell-bound CD16b antigen can be applied to similar GPI-linked antigens, for which purified antibody and fluorescent antibody against the same antigenic receptor are available.     

A multicolor,no-lyse no-wash assay for the absolute counting of CD34+ cells by flow cytometry

BACKGROUND: We previously developed a method for counting CD34(+) cells in unlysed whole blood. This method was applied to normal human bone marrow, peripheral blood after mobilization of progenitor cells, leukapheresis products, and cord blood and was validated with two different lyse-no wash methods. However, the main advantage that we described, erythrocyte discrimination using nucleic acid staining, was also the main restriction because additional markers for the immunologic characterization of CD34(+) cells cannot be included. METHODS: We used SYTO-13 and fluorescein isothiocyanate (FITC)-CD45 staining (FL1) instead of SYTO-13 and phycoerythrin (PE)Cy5-CD45 staining (FL3) to leave the third and fourth fluorescence parameters available for further characterization of CD34(+) cells. The new method was validated by applying it to cord blood samples (n = 20). RESULTS: FITC-CD45 antibody gave a 1.7-fold increase in mean fluorescence intensity over SYTO-13 alone. From absolute counts (CD34(+) cells per microliter), we plotted the differences between the methods against their mean, showing that differences fell into acceptable ranges. CONCLUSIONS: No-lyse procedures may represent an advance for cell immunophenotyping and it could be applied to the measurement of additional markers. Cytometry (Clin. Cytometry) 50:249-253, 2002.     

Cytofluorometric detection of rodent malaria parasites using red-excited fluorescent dyes

Flow cytometry is a potentially efficient approach for the quantification of parasitemias in experimental malaria infections and drug susceptibility assays using rodent malaria models such as Plasmodium berghei. In this study, we used two red DNA-binding fluorochromes, rhodamine 800 (R800) and LD700, to measure parasitemia levels in whole blood samples from mice infected with P. berghei. Blood samples were treated with RNAse A to eliminate RNA-derived signals. Propidium iodide, which stains both DNA and RNA, was used as a positive control. The parasitemia levels determined by R800 and LD700 were comparable to those calculated by microscopic analysis of blood smears and flow cytometry using Hoechst 33258. RNAse treatment did not affect these measurements. We also used R800 or LD700 to quantify parasitemias in mice infected with a GFP-expressing P. berghei line to correlate the parasitemia levels determined by DNA staining versus parasite numbers using GFP fluorescence as a surrogate measurement. A positive correlation was found between levels determined by flow cytometry using these dyes and those measured by GFP expression. Similar results were obtained when parasitemias determined by flow cytometry were compared to those determined by conventional microscopy. The limit of detection of infected red blood cells using R800 or LD700 staining was 0.1% and 0.15%, respectively. This study demonstrates that red laser-based flow cytometry using R800 or LD700 can be used for effective quantification of parasitemia levels in Plasmodium infected red blood cells. Furthermore, this method has the advantage that it does not require RNAse pretreatment and allows for a greater amount of cells to be analyzed for parasite burden than otherwise measured by conventional microscopy. ? 2011 International Society for Advancement of Cytometry.     

A small-volume technique for simultaneous immunophenotyping and apoptosis detection in rat whole blood by four-color flow cytometry

BACKGROUND: Cell permeabilization for the detection of intracellular molecules by flow cytometry is usually incompatible with whole blood. This article describes a new technique for the simultaneous detection of surface antigens and DNA content in rat whole blood. METHODS: In 20 microl of rat whole blood, DNA staining is obtained by permeabilization of cells using a standard red blood cell lysing reagent (Erythrolyse). Immunophenotyping and apoptosis detection by flow cytometry are achieved by using a combination of three surface markers (CD3, CD4, and CD8alpha) and a DNA binding dye (TO-PRO-3). RESULTS: After a 24-h incubation of whole blood with 1 microM dexamethasone, apoptotic lymphocytes were clearly distinguishable from normal lymphocytes by their reduced size and DNA content. The dexamethasone-induced percentage of apoptotic cells was 58.9 +/- 4.6 for CD4+ and 77.4 +/- 2.9 for CD8+ T cells, compared with 12.6 +/- 2.7 for CD4+ and 17.2 +/- 3.5 for CD8+ T cells in the absence of dexamethasone (data from 10 animals with duplicate samples). CONCLUSIONS: We have developed a new technique to permeabilize nucleated cells in microsamples of rat whole blood. The methodology allows simultaneous immunophenotyping and apoptosis detection in rat whole blood.     

Distinction between HLA class I-positive and -negative cervical tumor subpopulations by multiparameter DNA flow cytometry

BACKGROUND: The study of the molecular-genetic basis of heterogeneity of HLA class I expression in solid tumors is hampered by the lack of reliable rapid cell-by-cell isolation techniques. Hence, we studied the applicability of a flow cytometric approach (Corver et al.: Cytometry 2000;39;96-107). METHODS: Cells were isolated from five fresh cervical tumors and simultaneously stained for CD45 or vimentin (fluorescein isothiocyanate fluorescence), Keratin (R-phycoerythrin fluorescence), HLA class I (APC fluorescence), and DNA (propidium iodide fluorescence). A dual-laser flow cytometer was used for fluorescence analysis. Tissue sections from the corresponding tumors were stained for HLA class I antigens, keratin, vimentin, or CD45. RESULTS: Flow cytometry enabled the simultaneous measurement of normal stromal cells (vimentin positive), inflammatory cells (CD45 positive), epithelial cells (keratin positive), and DNA content readily. Normal stromal/inflammatory cells served as intrinsic HLA class I-positive as well as DNA-diploid references. Good DNA histogram quality was obtained (average coefficient of variation < 4%). Intratumor keratin positive subpopulations differing in HLA class I expression as well as DNA content could be clearly identified. Losses of allele-specific HLA class I expression found by immunohistochemistry were also detected by flow cytometry. CONCLUSIONS: We conclude that multiparameter DNA flow cytometry is a powerful tool to study loss of HLA class I expression in human cervical tumors. The method enables flow-sorting of discrete tumor and normal cell subpopulations for further molecular genetic analysis.     

Analyses of quality assessment studies using CD45 for gating lymphocytes for CD3(+)4(+)%

BACKGROUND: The purpose of this study was to assess whether laboratories which do not use CD45 for gating lymphocytes with three- (or four-) color flow cytometry (non-CD45 laboratories) for CD3(+)4(+)% and CD3(+)8(+)% do worse on quality assessment (QA) studies than laboratories which do use CD45 (CD45 laboratories). METHODS: Data came from blood specimens donated by 62 donors (50 HIV-positive) assayed over 2 years (November, 1996-October, 1998) by 35 laboratories in the NIAID DAIDS Flow Cytometry QA Program. RESULTS: Non-CD45 laboratories were significantly more likely to be classified as having unacceptable inter-laboratory results (far from the group median) than CD45 laboratories (5.6% vs 1.5%, P = 0.005 for CD3(+)4(+)%; 10.4% vs 5.0%, P = 0.007 for CD3(+)8(+)%). The intra-laboratory range of results on blinded replicates was significantly more likely to be deemed unacceptable (range >4%) in non-CD45 laboratories than in CD45 laboratories for CD3(+)8(+)% (14. 5% vs 3.5%, P = 0.002) but not for CD3(+)4(+)% (2.6% vs 1.5%, P = 0. 62). These differences in favor of CD45 gating were observed even though the non-CD45 laboratories had been doing three-color flow cytometry in the QA program significantly longer (P = 0.05) than the CD45 laboratories, and so would be expected to have fewer problems with the assay. CONCLUSIONS: Laboratories which choose to use a single CD3/CD4/CD8 tube for immunophenotyping may be sacrificing both accuracy and reproducibility.     

Iron Oxide Nanoparticle Assisted Purification and Mass Spectrometry Based Proteolytic Mapping of Intact CD4+T Cells from Human Blood

Abstract

Iron oxide nanoparticles have been used for many years as clinical applications. We have developed a rapid immunoaffinity isolation method of CD4⁺T cells from a mixed cell population of human blood using iron oxide nanoparticles. Anti CD4-antibody has been attached to iron oxide nanoparticles after its surface modification. The antibody tagged iron oxide nanoparticle beads are simply incubated with the mixed cell population of human blood and CD4⁺T cells are purified using an external magnetic field. The purification level was checked by fluorescence microscopy and flow cytometry. The purified CD4⁺T cells were digested with trypsin with different time periods and the products were analyzed by MALDI-TOF mass spectrometry, without further fractionation or purification, to obtain its proteome pattern. A database search showed a number of peptide masses matched specific to T-cell peptide masses. These results indicate that iron oxide nanoparticles are useful for CD4⁺T cell purification, and mass spectrometry based proteolytic fingerprint is simple and swift for identifying putative surface biomarkers from the whole cell surfaces.     

Flow cytometric analysis of macaque whole blood for antigen-specific intracellular cytokine production by T lymphocytes

We report here the standardized conditions for stimulation of macaque whole blood samples with various protein or peptide antigens, and production of significant intracellular levels of interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in CD4+ as well as CD8+ T lymphocytes. We observed significantly higher levels of TNF-alpha compared with IFN-gamma in both CD4+ and CD8+ T lymphocytes from all the macaque whole blood samples stimulated with staphylococcal enterotoxin B (SEB) as an antigen. Similarly, when whole blood samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine were stimulated with either the peptide cocktail or recombinant gp160, we observed production of significant levels of TNF-alpha by both CD4+ and CD8+ T lymphocytes. These results strongly support the utility of the whole blood cytokine flow cytometry methodology for determining antigen-specific immune responses of macaques in vaccine studies.     

Quantifying CD4 receptor protein in two human CD4+ lymphocyte preparations for quantitative flow cytometry

Background

In our previous study that characterized different human CD4+ lymphocyte preparations, it was found that both commercially available cryopreserved peripheral blood mononuclear cells (PBMC) and a commercially available lyophilized PBMC (Cyto-Trol™) preparation fulfilled a set of criteria for serving as biological calibrators for quantitative flow cytometry. However, the biomarker CD4 protein expression level measured for T helper cells from Cyto-Trol was about 16% lower than those for cryopreserved PBMC and fresh whole blood using flow cytometry and mass cytometry. A primary reason was hypothesized to be due to steric interference in anti- CD4 antibody binding to the smaller sized lyophilized control cells.

Method

Targeted multiple reaction monitoring (MRM) mass spectrometry (MS) is used to quantify the copy number of CD4 receptor protein per CD4+ lymphocyte. Scanning electron microscopy (SEM) is utilized to assist searching the underlying reasons for the observed difference in CD4 receptor copy number per cell determined by MRM MS and CD4 expression measured previously by flow cytometry.

Results

The copy number of CD4 receptor proteins on the surface of the CD4+ lymphocyte in cryopreserved PBMCs and in lyophilized control cells is determined to be (1.45 ± 0.09) × 10⁵ and (0.85 ± 0.11) × 10⁵, respectively, averaged over four signature peptides using MRM MS. In comparison with cryopreserved PBMCs, there are more variations in the CD4 copy number in lyophilized control cells determined based on each signature peptide. SEM images of CD4+ lymphocytes from lyophilized control cells are very different when compared to the CD4+ T cells from whole blood and cryopreserved PBMC.

Conclusion

Because of the lyophilization process applied to Cyto-Trol control cells, a lower CD4 density value, defined as the copy number of CD4 receptors per CD4+ lymphocyte, averaged over three different production lots is most likely explained by the loss of the CD4 receptors on damaged and/or broken microvilli where CD4 receptors reside. Steric hindrance of antibody binding and the association of CD4 receptors with other biomolecules likely contribute significantly to the nearly 50% lower CD4 receptor density value for cryopreserved PBMC determined from flow cytometry compared to the value obtained from MRM MS.

Electronic supplementary material

The online version of this article (doi:10.1186/1559-0275-11-43) contains supplementary material, which is available to authorized users.     

Changes in fluorescence intensity of selected leukocyte surface markers following fixation.

BACKGROUND: Immunophenotyping of blood leukocytes often involves fixation with paraformaldehyde prior to cytometry analysis. However, the influence of cell type and marker specificity on the stability of fluorescence intensity after fixation has not been well studied. METHODS: Human whole blood was stained using a panel of fluorescein isothiocyanate-labeled antibodies to surface markers. Unfixed and fixed samples were analyzed by flow cytometry at 0, 2, 4, 6, 24, 48, and 96 h after staining. Fluorescence measurements were converted to molecules of equivalent soluble fluorochrome for comparison. RESULTS: Fixation caused a significant decrease in both forward and side scatter at 48 h which required gating adjustments to achieve resolution of cell populations. The autofluorescence increased progressively in fixed samples (ninefold at 96 h for monocytes). Variable decreases in marker-associated fluorescence became apparent after correction for autofluorescence. The magnitude of the decrease at 96 h varied with cell type and marker, from 5% for CD32 on monocytes to 39% for CD16 on neutrophils. CONCLUSION: The change in fluorescence intensity following staining and fixation of leukocytes varies with cell type and surface marker. Fluorescence stability should be determined for each cell type and marker used, and the confounding effects of fixation on cell autofluorescence should be considered.     

Cytometry and flow cytometry of 4',6-diamidino-2-phenylindole (DAPI)-stained suspensions of nuclei released from fresh and fixed tissues of plants

Cytometry and flow cytometry were used to study characteristics of fluorescence of the DNA-DAPI complex in nuclei released from different fresh and formaldehyde-fixed pea ( Pisum sativum L. cv. Lincoln) tissues. The two methods of isolation are compared and discussed as well as their possible use for quantitative analysis of DNA in plant tissues. With fixed tissues it is possible to obtain a number of nuclei sufficient for the flow cytometric analysis, even using small amounts of plant tissue.     

Deconvoluting post-transplant immunity: cell subset-specific mapping reveals pathways for activation and expansion of memory T, monocytes and B cells

A major challenge for the field of transplantation is the lack of understanding of genomic and molecular drivers of early post-transplant immunity. The early immune response creates a complex milieu that determines the course of ensuing immune events and the ultimate outcome of the transplant. The objective of the current study was to mechanistically deconvolute the early immune response by purifying and profiling the constituent cell subsets of the peripheral blood. We employed genome-wide profiling of whole blood and purified CD4, CD8, B cells and monocytes in tandem with high-throughput laser-scanning cytometry in 10 kidney transplants sampled serially pre-transplant, 1, 2, 4, 8 and 12 weeks. Cytometry confirmed early cell subset depletion by antibody induction and immunosuppression. Multiple markers revealed the activation and proliferative expansion of CD45RO(+)CD62L(-) effector memory CD4/CD8 T cells as well as progressive activation of monocytes and B cells. Next, we mechanistically deconvoluted early post-transplant immunity by serial monitoring of whole blood using DNA microarrays. Parallel analysis of cell subset-specific gene expression revealed a unique spectrum of time-dependent changes and functional pathways. Gene expression profiling results were validated with 157 different probesets matching all 65 antigens detected by cytometry. Thus, serial blood cell monitoring reflects the profound changes in blood cell composition and immune activation early post-transplant. Each cell subset reveals distinct pathways and functional programs. These changes illuminate a complex, early phase of immunity and inflammation that includes activation and proliferative expansion of the memory effector and regulatory cells that may determine the phenotype and outcome of the kidney transplant.     

CD45-assisted PanLeucogating for accurate,cost-effective dual-platform CD4+ T-cell enumeration

BACKGROUND: North American and European guidelines for dual-platform (DP) flow cytometry recommend absolute CD4 T-cell counts to be calculated from two parameters: the absolute lymphocyte counts obtained on a hematology analyzer and the percentages of CD4+ cells among lymphocytes (CD4%/lympho) obtained by flow cytometry. Nevertheless, the identification of lymphocytes is error-prone: a poor match between these common denominators in the two systems is the main source of inaccuracy. In contrast, total leucocyte counts (white cell counts [WCC]) and CD4% among the gated CD45+ leucocytes (CD4%/leuco) can be determined with greater accuracy. METHODS: We introduced "PanLeucogating," i.e., we used total leucocytes as the common denominator for improving the precision of DP absolute CD4 counting. Correlations and Bland-Altman tests were used for statistical analysis. RESULTS: First, 22 stabilized blood product samples were provided by U.K. National External Quality Assessment Scheme (NEQAS) and a higher accuracy and precision of CD4 counts were documented using PanLeucogating compared with lymphocyte gating. Next, 183 fresh and 112 fixed (TransFix) whole blood samples were used to compare DP methods and single-platform (SP) methodology, including both volumetric and bead-based techniques. A particularly high correlation and comparable precision of absolute CD4 counts were observed between the SP volumetric method and DP PanLeucogating (R(2) = 0.990; bias 6 +/- SD 17%). The SP volumetric method showed lower levels of agreement with the DP lymphocyte gating (R(2) = 0.758; bias 14 +/- SD 51%) and with the SP bead-based method (R(2) = 0.923; bias 4 +/-SD 31%). CONCLUSIONS: These observations show that DP leucocyte counts (WCC) should replace lymphocyte counts as the "common denominator" although CD4%/lympho values can, as an extra step, be also provided readily if requested. When coupled with quality control for WCC on hematology analyzers, the DP method with CD45 PanLeucogating represents a robust CD4 T-cell assay that is as accurate as the SP volumetric technique. This DP method uses only two, CD45 and CD4, antibody reagents and can be run on any pair of hematological analyzer plus flow cytometer.     

Influence of EDTA and heparin on lipopolysaccharide binding and cell activation, evaluated at single-cell level in whole blood

BACKGROUND: The use of whole blood (WB) in studying lipopolysaccharide (LPS)-induced cellular activation preserves the milieu in which LPS-cell interaction occurs in vivo. However, little information is available on using such a system at a single-cell level. We evaluated LPS binding and cell activation in WB by using flow cytometry. The influence of heparin or EDTA as anticoagulants was also addressed. METHODS: Blood was obtained from healthy donors in EDTA and/or heparin tubes. Biotinylated LPS (LPSb) was used to evaluate cell binding of LPS in WB. Cells were surface stained with appropriate antibodies and LPSb was detected by adding streptavidin-allophycocyanin (APC). LPS-induced activation was evaluated by the expression of surface activation markers and by the detection of intracellular tumor necrosis factor-alpha (TNF-alpha). RESULTS: LPSb bound promptly to monocytes in EDTA- and heparin-treated blood. In EDTA-treated blood, membrane-bound LPSb decreased after 60 min of incubation, whereas it remained detectable in heparinized blood during the 6 h of incubation. LPS induced TNF-alpha and enhanced the expression of HLA-DR in monocytes, as well as the expression of CD69 in T and B lymphocytes. Induction of both TNF-alpha in monocytes and CD69 in lymphocytes was more efficient in heparinized blood. CONCLUSION: Detection of membrane-bound LPSb on monocytes differed in EDTA or heparin-treated blood, and cell activation was better obtained in heparinized blood.     

Automated four-color analysis of leukocytes by scanning fluorescence microscopy using quantum dots.

BACKGROUND: Scanning fluorescence microscope (SFM) is a new technique for automated motorized microscopes to measure multiple fluorochrome labeled cells (Bocsi et al., Cytometry A 2004, 61:1-8). AIMS: We developed a four-color staining protocol (DNA, CD3, CD4, and CD8) for the lymphocyte phenotyping by SFM. METHODS: Organic (Alexa488, FITC, PE-Alexa610, CyChrom, APC) and inorganic (quantum dot (QD) 605 or 655) fluorochromes were used and compared in different combinations. Measurements were performed in suspension by flow cytometer (FCM) and on slide by SFM. RESULTS: Both QDs were detectable by the appropriate Axioplan-2 and FCM filters and the AxioCam BW-camera. CD4/CD8 ratios were highly correlated (P = 0.01) between the SFM and FCM. CONCLUSION: Automated SFM is an applicable tool for CD4/CD8 ratio determination in peripheral blood samples with QDs.     

Multisite comparison of methods for the quantitation of the surface expression of CD38 on CD8(+) T lymphocytes. The ACTG Advanced Flow Cytometry Focus Group

We evaluated the effect of specimen processing variations and quantitation methods on quantitative determination of CD38 expression on CD8 T lymphocytes. Neither lysing reagent (ammonium chloride versus BD FACSlyse), fixation (paraformaldehyde versus no final fixation step), nor acquisition delay (acquisition within 6 h after fixation versus 24 h after fixation) had a significant effect on CD38 relative fluorescent intensity or CD38 quantitative estimates (RFI or antibodies bound per cell). The only significant difference in fluorescent intensity and CD38 antibodies bound per cell (ABC) was encountered when whole blood was held for 24 h prior to staining and fixation and then acquired after another 24-h hold. However, for all sample processing methods above, the CD4 biologic calibrator and QuantiBRITE bead methods gave significantly different estimates of CD38 intensity. In many cases, however, these differences are relatively small and were more pronounced in certain laboratories. We conclude that there is some flexibility in sample processing methods for quantitative CD38 determination; however, it is preferable for a laboratory to employ one method of fluorescence quantitation calculation consistently because small differences are detected between different methods. Cytometry (Comm. Clin. Cytometry) 42:174-179, 2000.     

Flow cytometric, phase-resolved fluorescence measurement of propidium iodide uptake in macrophages containing phagocytized fluorescent microspheres

BACKGROUND: Spectral interference (overlap) from phagocytosed green-yellow (GY) microspheres in the flow cytometric, red fluorescence emission measurement channel causes errors in quantifying damaged/dead alveolar macrophages by uptake of propidium iodide. METHODS: Particle burdens of uniform GY fluorescent microspheres phagocytosed by rat alveolar macrophages and the discrimination of damaged/dead cells as indexed by propidium iodide uptake were assessed with conventional and phase-sensitive flow cytometry. RESULTS: The fluorescence spectral emission from phagocytosed microspheres partly overlapped the propidium iodide red fluorescence emission and interfered with the measurement of damaged/dead cells when using conventional flow cytometry without subtractive compensation. This caused errors when estimating the percentage of nonviable, propidium iodide-positive, phagocytic macrophages. The interference was eliminated by employing phase-sensitive detection in the red fluorescence measurement channel based on differences in fluorescence lifetimes between the fluorescent microspheres and propidium iodide. Intrinsic cellular autofluorescence, whose fluorescence lifetime is approximately the same as that of the phagocytosed microspheres, also was eliminated in the phase-sensitive detection process. Because there was no detectable spectral interference of propidium iodide in the green fluorescence (phagocytosis) measurement channel, conventional fluorescence detection was employed. CONCLUSIONS: Phase-resolved, red fluorescence emission measurement eliminates spectral overlap errors caused by autofluorescent phagocytes that contain fluorescent microspheres in the analyses of propidium iodide uptake.Cytometry 39:45-55, 2000. Published 2000 Wiley-Liss, Inc.