Method For Flow Cytometric Analysis Research Articles

An alternative processing approach to increase CD138 intensity in flow cytometric analysis of plasma cells.

Surface median immunofluorescence intensity (MFI) of plasma cells antigens, particularly CD138, by flow cytometry underestimates plasma cell populations when compared with that estimated by morphological assessment on Wright's-stained slides. CD138 MFI using traditional sample preparation methods for flow cytometric analysis is often dim and difficult to interpret due to multiple factors. This becomes critical when diagnosing and accurately classifying plasma cell dyscrasias. In this study, we analyzed 280 flow cytometric results collected from 2016 to 2022 for CD38 and CD138 MFI on bone marrow aspirates performed by two different methods of sample processing-traditional method of lyse-wash and the alternative method of lyse-no-wash. Visual examination of histograms showed a clear advantage to CD138 expression intensity with the no-wash method. Although no significant difference was observed in CD38 MFI between the two techniques (p = 0.3), considerable improvement was observed in CD138 MFI with the lyse-no-wash technique of sample processing compared with the conventional method (p = 0.003). We concluded that the method of lyse-no-wash is superior to traditional methods especially when it comes to handling bone marrow aspirate samples for plasma cell immunophenotyping. This alternate technique increases the sensitivity of flow cytometry to detect plasma cells resulting in bright and crisp signal intensity for surface CD138. This technique may be particularly advantageous when analyzing low tumor burden such as minimal residual disease.

MTOR-C/EBPβ-c-Myc Axis Regulates the Behavior of Myeloid-Biased Hematopoietic Stem Cells Under Stress Conditions

In stress or regenerative conditions, hematopoietic stem cell (HSC) population drastically alters its behavior; they rapidly enter into cell cycle, differentiate to multipotent progenitors (MPPs) at the expense of self-renewal activity, and are reprogrammed toward predominantly myeloid-biased hematopoiesis. In these processes, essential roles of myeloid transcription factors in regenerating HSCs are anticipated. C/EBPβ regulates both proliferation and differentiation of granulocytic precursors in stress conditions. However, its functions in HSCs during stresses are largely unknown.In the past several years, we have been investigating the roles of C/EBPβ in HSCs under stress conditions, especially focusing on the expression pattern of C/EBPβ isoforms and their respective functions. Cebpb is a unique single exon gene, from which two long protein isoforms (LAP* and LAP) and one truncated isoform lacking N-terminal activating domain (LIP) are translated by different usage of initiating codons. To monitor the expression pattern of these isoforms in scarce cells, we have devised an intracellular double staining method for flow cytometric analysis using two distinct anti-C/EBPβ antibodies recognizing N- and C-terminus, respectively (57th ASH annual meeting, #3580).Here, we refined this method to reveal the characteristic expression pattern of C/EBPβ isoforms in HSCs in vivo in response to stresses. In regenerative conditions after either 5-FU treatment or transplantation, we detected remarkable LIP-dominant upregulation in LT-HSCs at early phases, which was followed by increase of LAP*/LAP at later phases. This stress-induced translational upregulation and alteration of C/EBPβ were significantly suppressed by short-term administration of rapamycin, a selective inhibitor of mTOR, suggesting its dependence on mTORC1 pathway at least in part.We further found that the stress-induced LIP-dominant upregulation of C/EBPβ was remarkable in LT-HSCs with higher surface CD150 expression (CD150high LT-HSCs), which possess myeloid-biased potential. Cell cycle analyses at early phases after 5-FU treatment, when LIP was predominantly expressed, revealed that CD150high HSCs were significantly less quiescent than CD150low HSCs but this difference was abolished in Cebpb-/- HSCs, suggesting that C/EBPβ specifically activated the cell cycle of CD150high “myeloid-biased” HSCs.We next examined the functions of C/EBPβ isoforms in HSCs in vivo . We transplanted WT BM cells retrovirally transduced with each C/EBPβ isoform into sublethally irradiated WT mice, and then analyzed the transduced cells in the recipients. LAP*- or LAP-transduced cells disappeared as early as 3 weeks after transplantation, probably due to their rapid myeloid differentiation. LIP-transduced HSCs were significantly less quiescent, exhibited more accelerated differentiation to MPPs, especially lymphoid-primed MPP4s, and produced more lymphoid-biased outputs. These results indicate that LIP induces cell cycle activation of HSCs and expansion of MPP pool, while antagonizing the other long isoforms that induce rapid myeloid commitment and premature exhaustion of HSCs.As a candidate for the downstream target of C/EBPβ, especially LIP, in HSCs, we focused on Myc (encoding c-Myc). LIP-transduced EML cells, a mouse HSC line, expressed 1.5-fold higher level of Myc mRNA than those transduced with control, and LIP-transduced HSCs in vivo expressed 2-fold higher than control. We next compared Myc mRNA expression between WT and Cebpb-/- HSCs at early phases of regeneration post 5-FU treatment and transplantation, when LIP was predominantly upregulated. Then, we found that significantly lower level of Myc transcripts was expressed in Cebpb-/- HSCs. When we performed a ChIP-qPCR analysis using C/EBPβ-transduced EML cells, remarkable enrichment of C/EBPβ was observed at the distal enhancer of Myc gene. These data suggest that both forced overexpression and physiological elevation of LIP positively regulate Myc transcription in HSCs likely through direct binding to Myc enhancer.In summary, our findings illustrate that stress-induced upregulation and alteration of C/EBPβ protein, mediated by mTORC1 and modulating Myc transcription, is significantly involved in stress-mode behavior of HSCs especially with myeloid bias. DisclosuresNo relevant conflicts of interest to declare.

Flow Cytometric Analyses of p53-Mediated Cell Cycle Arrest and Apoptosis in Cancer Cells.

Phenotypic analysis of the effects of a gene of interest may be limited because stable expression of some genes leads to adverse consequences in cell survival, such as disturbance of cell cycle progression, senescence, autophagy, and programmed cell death. One of the best examples is tumor suppressor p53. p53 functions as a tumor suppressor by inducing cell cycle arrest and apoptosis in response to genotoxic and environmental insults. The choice and timing of either pathways induced by p53 depend on cellular context, cell types, and the degree of cellular/genomic damage (For review, see (Chen J, Cold Spring Harb Perspect Med 6:a026104, 2016)). The uncertainty makes the studies on the long-term effects of p53 in cells challenging. This chapter describes a method of flow cytometric analysis of ectopic expression of p53 to better quantify cell cycle distribution and apoptosis in cells treated with DNA damaging agents. The method can be easily adapted to other genes of interest to study their contributions to the fate of variety of cell types in response to endogenous or exogenous stresses.

C/EBPβ Isoforms Regulate Proliferation and Differentiation of Regenerating Hematopoietic Stem/Progenitor Cells

Under stress or regenerative conditions, HSCs rapidly enter into cell cycle and are reprogrammed toward myeloid-biased hematopoiesis to meet the increasing demand of myeloid cells. We have previously shown that the transcription factor C/EBPβ plays critical roles at the level of HSPCs under stress conditions (Nat Immunol 2006, J Immunol 2012, Leukemia 2013 and Blood Adv 2019). However, underlying molecular mechanisms of action remain largely unknown. In this study, we have investigated the detailed function of C/EBPβ in regulation of HSPCs. We first evaluated the impact of C/EBPβ on the cell cycle status of LT-HSCs. To exclude the cell-extrinsic contribution of C/EBPβ, CD45.2+ BM cells from WT or Cebpb-/- mice were transplanted into lethally irradiated CD45.1+ WT mice, and these "BM-replaced" recipients were subjected to the following experiments. At steady state, the cell cycle statuses and the numbers of HSPCs did not significantly differ between the recipients of WT cells and those of Cebpb-/- cells. Immediately after 5-FU treatment, WT LT-HSCs entered the cell cycle, as revealed by the decreased percentage of cells in G0 phase and the increased percentage of cells in S/G2M phase. All these parameters of cell cycle acceleration were observed prior to the nadir of LT-HSCs induced by 5-FU and were significantly attenuated in Cebpb-/- LT-HSCs. Next, we assessed the numbers of LT-HSCs, KSL cells, and KL cells after 5-FU treatment. Following the nadir, the recovery of LT-HSCs preceded that of KSL and KL cells, suggesting the differentiation of LT-HSCs to KSL and KL cells. In the recipients of Cebpb-/- cells, the recovery of KSL and KL cells was delayed significantly. Collectively, cell cycle acceleration and subsequent differentiation of LT-HSCs under stress conditions were impaired in the absence of Cebpb. The Cebpb is a single exon gene, and three isoforms, namely, LAP*, LAP and LIP which lacks N-terminus, are translated from its unique mRNA. Due to their structural difference, they should have distinct functions. Here, we focused on expression and functions of these isoforms in regenerating HSPCs. To monitor expression of these isoforms in small numbers of HSCs, we devised a novel intracellular double staining method for flow cytometric analysis using two distinct anti-C/EBPβ antibodies. An antibody against the C-terminus of C/EBPβ recognized all three isoforms, while an antibody against the N-terminus of C/EBPβ only recognized LAP* and LAP. Thus, simultaneous staining with both antibodies should enable us to distinguish cells that dominantly expressed LIP (C-term+ N-term-) from those that expressed all three isoforms (C-term+ N-term+). Using this method, we monitored the expression patterns of these isoforms in LT-HSCs after 5-FU treatment. LT-HSCs initially became C-term single positive in response to 5-FU and subsequently changed to C- and N-term double positive, suggesting that LIP was upregulated prior to LAP/LAP* under stress conditions. These results suggest that phase-specific upregulation of LIP and LAP/LAP* is strongly associated with phase-specific functions of C/EBPβ in cell cycle activation and differentiation, respectively. Indeed, when EML cells, a mouse HSC line, were retrovirally transduced with LIP, the transduced cells were more proliferative and actively cycling than those transduced with the control vector, whereas proliferation and cell cycle were markedly suppressed in LAP*- and LAP-expressing EML cells. LIP-expressing cells remained undifferentiated, while LAP*- and LAP-expressing cells rapidly differentiated into CD11b+ myeloid cells and eventually stopped proliferating. In summary, our findings clearly suggest that sequential upregulation of C/EBPβ isoforms is critical for the regulation of HSCs under stress conditions. LIP amplifies the "reservoir" of HSPCs by accelerating the proliferation of HSCs during the early phase of regeneration, while LAP*/LAP induce their myeloid differentiation at a later phase. These findings should facilitate our understanding of the pathophysiology of infection, inflammation, and regenerating hematopoiesis in response to myeloablative chemotherapies or hematopoietic stem cell transplantation, all of which increase the hematopoietic demands. Disclosures Hirai: Kyowa Kirin: Research Funding.

A field-applicable method for flow cytometric analysis of granulocyte activation: Cryopreservation of fixed granulocytes.

Upon activation granulocytes upregulate several adhesion molecules (CD11b) and granule proteins (CD35, CD66b) and shed surface l-selectin (CD62L). These changes in expression, as assessed by flow cytometry, can be used as markers for activation. Whereas these markers are usually studied in fresh blood samples, a new method is required when samples are collected at a field site with no direct access to a flow cytometer. Therefore, we developed and tested a field-applicable method in which fixed leukocytes were cryopreserved. Using this method, the intensity of granulocyte activation markers was compared to samples that were either stained fresh, or fixed prior to staining but not cryopreserved. In addition, the response to an in vitro stimulation with fMLF was determined. While we observed differences in marker intensities when comparing fresh and fixed granulocytes, similar intensities were found between fixed cells that had been cryopreserved and fixed cells that did not undergo cryopreservation. Although fixation using FACS lysing solution might lead to membrane permeabilization, activation markers, and the responsiveness to fMLF or eotaxin could still be clearly measured. This method will, therefore, enable future studies of granulocyte activation in settings with limited resources and will allow simultaneous analysis of samples collected at different time points. © 2018 International Society for Advancement of Cytometry.

A Fast, Easy, and Customizable Eight-Color Flow Cytometric Method for Analysis of the Cellular Content of Bronchoalveolar Lavage Fluid in the Mouse.

The cell composition of bronchoalveolar lavage fluid (BAL) is an important indicator of airway inflammation. It is commonly determined by cytocentrifuging leukocytes on slides, then staining, identifying, and counting them as eosinophils, neutrophils, macrophages, or lymphocytes according to morphological criteria under light microscopy, where it is not always easy to distinguish macrophages from lymphocytes. We describe here a one-step, easy-to-use, and easy-to-customize 8-color flow cytometric method for performing differential cell count and comparing it to morphological counts on stained cytospins. This method identifies BAL cells by a simultaneous one-step immunolabeling procedure using antibodies to identify T cells, B cells, neutrophils, eosinophils, and macrophages. Morphological analysis of flow-sorted cell subsets is used to validate this protocol. An important advantage of this basic flow cytometry protocol is the ability to customize it by the addition of antibodies to study receptor expression at leukocyte cell surfaces and identify subclasses of inflammatory cells as needed. © 2017 by John Wiley & Sons, Inc.

A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflamed Murine Non-Lymphoid Tissues.

Flow cytometry is used extensively to examine immune cells in non-lymphoid tissues. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. We developed a protocol for the flow cytometric analysis of non-lymphoid tissues, including methods of tissue preparation, a 10-fluorochrome panel for cell staining, and a standardized gating strategy, that allows the simultaneous identification and quantification of all major immune cell types in a variety of normal and inflamed non-lymphoid tissues. We demonstrate that our basic protocol minimizes cell loss, reliably distinguishes macrophages from dendritic cells (DC), and identifies all major granulocytic and mononuclear phagocytic cell types. This protocol is able to accurately quantify 11 distinct immune cell types, including T cells, B cells, NK cells, neutrophils, eosinophils, inflammatory monocytes, resident monocytes, alveolar macrophages, resident/interstitial macrophages, CD11b- DC, and CD11b+ DC, in normal lung, heart, liver, kidney, intestine, skin, eyes, and mammary gland. We also characterized the expression patterns of several commonly used myeloid and macrophage markers. This basic protocol can be expanded to identify additional cell types such as mast cells, basophils, and plasmacytoid DC, or perform detailed phenotyping of specific cell types. In examining models of primary and metastatic mammary tumors, this protocol allowed the identification of several distinct tumor associated macrophage phenotypes, the appearance of which was highly specific to individual tumor cell lines. This protocol provides a valuable tool to examine immune cell repertoires and follow immune responses in a wide variety of tissues and experimental conditions.

Flow Cytometry Analysis of Thymic Epithelial Cells and Their Subpopulations.

The parenchyma of the thymus is compartmentalized into the cortex and the medulla, which are constructed by cortical thymic epithelial cells (cortical TECs, cTECs) and medullary thymic epithelial cells (mTECs), respectively. cTECs and mTECs essentially and differentially regulate the development and repertoire selection of T cells. Consequently, the biology of T cell development and selection includes the study of TECs in addition to the study of developing T cells and other hematopoietic cells including dendritic cells. In this chapter, we describe the methods for flow cytometric analysis and sorting of TECs and their subpopulations, including cTECs and mTECs.

A Novel Lung Explant Model for the Ex Vivo Study of Efficacy and Mechanisms of Anti-Influenza Drugs

Influenza A virus causes considerable morbidity and mortality largely because of a lack of effective antiviral drugs. Viral neuraminidase inhibitors, which inhibit viral release from the infected cell, are currently the only approved drugs for influenza, but have recently been shown to be less effective than previously thought. Growing resistance to therapies that target viral proteins has led to increased urgency in the search for novel anti-influenza compounds. However, discovery and development of new drugs have been restricted because of differences in susceptibility to influenza between animal models and humans and a lack of translation between cell culture and in vivo measures of efficacy. To circumvent these limitations, we developed an experimental approach based on ex vivo infection of human bronchial tissue explants and optimized a method of flow cytometric analysis to directly quantify infection rates in bronchial epithelial tissues. This allowed testing of the effectiveness of TVB024, a vATPase inhibitor that inhibits viral replication rather than virus release, and to compare efficacy with the current frontline neuraminidase inhibitor, oseltamivir. The study showed that the vATPase inhibitor completely abrogated epithelial cell infection, virus shedding, and the associated induction of proinflammatory mediators, whereas oseltamivir was only partially effective at reducing these mediators and ineffective against innate responses. We propose, therefore, that this explant model could be used to predict the efficacy of novel anti-influenza compounds targeting diverse stages of the viral replication cycle, thereby complementing animal models and facilitating progression of new drugs into clinical trials.

Fine-Needle Aspiration is Superior to Needle Core Biopsy as a Sample Aquisition Method for Flow Cytometric Analysis in Suspected Hematologic Neoplasms.

Background: Common minimally invasive methods for acquiring samples for flow cytometric immunophenotyping (FCI) include fine needle aspiration (FNA) and needle core biopsy (NCB). FCI requires a sufficient quantity of viable cells for adequate evaluation. Methods: We collected patient data from our files of all FCI cases sampled via FNA or NCB from 1/1/03 and 6/1/12. Total Viable Cells (TVC) was calculated by multiplying the volume, viability and concentration and then converted to logarithmic scale as "Log TVC." Statistical analysis was performed using SPSS. Results: 571 FCI cases at our institution were reviewed covering the period from 2003 to 2012 and 456 total cases were analyzed. 116 cases were sampled by NCB and 340 were sampled by FNA. Comparing FNA to NCB subgroups demonstrated FNA to be superior in mean specimen viability, TVC, and cases with a final FCI interpretation. The cellularity of the sample (in Log TVC) correlates with the likelihood of achieving a FCI interpretation. The point where at least 50% of cases have a diagnostic FCI interpretation occurs between Log TVC of 5.0 - 5.25. However, FNA based cases had a higher proportion of samples with an indeterminate final diagnosis. Conclusions: FNA was found to be significantly superior to NCB in obtaining material for FCI. However, NCB resulted in fewer indeterminate final diagnoses due to benefit of histologic correlation. In our opinion, NCB for histology combined with dedicated FNA material for FCI may yield the best results for a minimally invasive approach to the diagnosis of hematologic neoplasms. © 2014 Clinical Cytometry Society.

Methods for Flow Cytometric Analysis of T Cell Subsets in HIV-infected Patients: 2-Color versus 4-Color

Methods for Flow Cytometric Analysis of T Cell Subsets in HIV-infected Patients: 2-Color versus 4-Color

A heparin-based method for flow cytometric analysis of microparticles directly from platelet-poor plasma in calcium containing buffer

BackgroundCharacterization of circulating microparticles (MPs) is usually performed by flow cytometry. Annexin V, a protein that Ca2+-dependently binds to phosphatidylserine, has been used to define entire microparticle (MP) populations, but not all MPs bind AnxV. Recent reports have correlated AnxV negative MPs to clinical parameters in systemic diseases, which emphasize the importance of including characterization of AnxV-binding. An obstacle in flow cytometric analysis of AnxV-binding to MPs is that plasma may clot when adding the Ca2+-containing buffers. We here devise a simple method for comprehensive assessment of circulating MPs directly from platelet-poor plasma with characterization of AnxV-binding and of cellular origin of MPs. Materials and methodsWith 49 samples (20 healthy controls and 29 SLE patients) a flow cytometric method analyzing MPs directly from platelet-poor plasma was developed and compared with an established, more laborious method. The method relies on using heparin to inhibit plasma coagulation induced by Ca2+ and subsequent incubation with labeling reagents directly in platelet-poor plasma. ResultsIn comparison with standard methods the new direct method showed low variability (1–12% in total MP measurement), had higher MP counts (i.e. prevents loss of MPs), was less time consuming (saved 2/3 of sample processing time) and results correlated well with an established method of analysis of washed MPs by flow cytometry (r>0.7; p<0.0001). Additionally heparin was shown not to impact MP counts, longtime storage did not alter MP concentrations, and distinct effects of freezing on MP characteristics were confirmed. ConclusionsThe direct method reduces variability due its simplicity and faster handling, and it saves sample material. It is a convenient, fast, and reproducible method for assessing the population of circulating MPs and correlates well with more cumbersome approaches. These benefits make the method well suited for large studies.

Glycerol-treated nuclear suspensions—an efficient preservation method for flow cytometric analysis of plant samples

Flow cytometry (FCM) has been widely used in plant science to determine the amount of nuclear DNA, either in absolute units or in relative terms, as an indicator of ploidy. The requirement for fresh material in some applications, however, limits the value of FCM in field research, including plant biosystematics, ecology and population biology. Dried plant samples have proven to be a suitable alternative in some cases (large-scale ploidy screening) although tissue dehydration is often associated with a decrease in the quality of FCM analysis. The present study tested, using time-scale laboratory and in situ field experiments, the applicability of glycerol-treated nuclear suspension for DNA flow cytometry. We demonstrate that plant nuclei preserved in ice-cold buffer + glycerol solution remain intact for at least a few weeks and provide estimates of nuclear DNA content that are highly comparable and of similar quality to those obtained from fresh tissue. The protocol is compatible with both DAPI and propidium iodide staining, and allows not only the determination of ploidy level but also genome size in absolute units. Despite its higher laboriousness, glycerol-preserved nuclei apparently represent the most reliable way of sample preservation for genome size research. We assume that the protocol will provide a vital alternative to other preservation methods, especially when stringent criteria on the quality of FCM analysis are required.

Medicaments: Gifts from the jungle

Medicaments: Gifts from the jungle

Relationship between the phenotypes and functions of peripheral blood dendritic cells and the different spleen deficiency syndrome types in patients with chronic hepatitis B

To study the phenotypes and functions of dendritic cells (DCs) derived from peripheral blood monocytes of chronic hepatitis B (CHB) patients with different traditional Chinese medicine (TCM) syndrome types, and to explore the relationship between TCM syndrome type and DC functions. Sixty CHB patients were included in this study. All the CHB patients were divided into spleen deficiency and liver stagnation, spleen deficiency and dampness-heat and deficiency of both spleen and kidney groups according to TCM syndrome diagnosis standard. There were 20 cases in each group, and ten healthy people were included as normal control. The volunteer's peripheral blood was collected for monocyte separation, biochemical test and hepatitis B virus DNA loads detection. DCs were induced and isolated from peripheral blood monocytes, and then the expressions of surface markers CD80, CD86, CD1a and HLA-DR were detected by flow cytometric analysis method. Interleukin-10 (IL-10) production of the DCs was quantified by enzyme-linked immunosorbent assay. The proliferation of DCs in the CHB patients was slower than that in the healthy volunteers (P<0.05). The expressions of DC surface molecules such as CD80, CD86, and CD1a were obviously decreased in the CHB patients as compared with those in the healthy volunteers (P<0.05). More over, expressions of DC surface molecules were different among CHB patients with different TCM syndrome types. The positive expressions of CD80, CD1a, and HLA-DR in the CHB patients with spleen deficiency and liver stagnation were obviously higher than those in the CHB patients with deficiency of both spleen and kidney (P<0.05), and the CD1a expression in the CHB patients with spleen deficiency and dampness-heat was higher than that in the CHB patients with deficiency of both spleen and kidney (P<0.05). In DC culture supernatant, the IL-10 concentration of the CHB patients with deficiency of both spleen and kidney was higher than that of the CHB patients with spleen deficiency and liver stagnation (P<0.05), and the IL-10 concentrations of the CHB patients with different TCM syndrome types were higher than that of the healthy volunteers (P<0.05). During the pathogenic course of CHB, the phenotypes and functions of DCs are different in CHB patients with different TCM syndrome types. It suggests that there is a correlation between TCM syndrome type and body immunity function.

Antigen-specific B cell detection reagents: use and quality control.

Tests for immunoglobulin reactivity with specific antigens are some of the oldest and most used assays in immunology. With efforts to understand B cell development, B cell dysregulation in autoimmunity, and to generate B cell vaccines for infectious agents, investigators have found the need to understand the ontogeny and regulation of epitope-specific B cell responses. The synchrony between surface and secreted antibodies for individual B cells has led to the development of reagents and techniques to identify antigen-specific B cells via reagent interactions with the B cell receptor complex. B cell antigen-specific reagents have been reported for model systems of haptens, for whole proteins, and for identification of double stranded (ds) DNA antibody-producing B cells using peptide mimics. Here we provide an overview of reported techniques for the detection of antigen-specific B cell responses via secreted antibody or by the surface B cell receptor and briefly discuss our recent work developing a panel of reagents to probe the B cell response to HIV-1 envelope. We also present an analysis of strengths and weaknesses of various methods for flow cytometric analysis of antigen-specific B cells.

Platelets in the paediatric population: the influence of age and the limitations of automation

Accurate and precise platelet counting is important for the clinical management of children with platelet disorders. Current automated technologies are often unable to discriminate platelets from non-platelet particles particularly in circumstances where platelet anisocytosis is common. This study compares manual methodology and the automated technologies; impedance, optical density and CD61 immunoplatelet method (available on the Cell Dyn 4000) with the reference method of flow cytometric analysis in a paediatric population. A total of 141 samples were analysed and divided into specific age related groups and groups with thrombocytopenia and thrombocytosis. Data analysis showed that the CD61 method compared best with the reference method and this was evident in all the specified groups. The mean platelet count obtained by optical and manual methods were lower, suggesting that these methods are less reliable. The impedance count method was accurate despite its limitations. Strong correlations were observed in the 2-14 year age group but there was greater variation in the <1 month group supporting the theory that there is a greater variation in platelet characteristics in neonates. The CD61 method is the automated method of choice and would be particularly useful in the problem groups (platelet counts <50 x 10(9)/l and neonates <1 month old).

An efficient method for flow cytometric analysis of pollen and detection of 2n nuclei in Brassica napus pollen.

A simple and reliable method was developed for isolating pollen nuclei from Brassica napus and Triticum aestivum for DNA analysis using flow cytometry. The nuclei were released from pollen by ultrasonic treatment. The isolated nuclei following filtration through nylon mesh and a purification procedure were suitable for flow cytometric analysis as well as for isolating genomic DNA. Ultrasonic treatment time was optimized for B. napus pollen at different developmental stages. The method is effective and suitable for the preparation of many samples. We analyzed the nuclear DNA levels in pollen of B. napus at three major developmental stages as well as in mature wheat pollen. Only a single 1C peak representing the haploid DNA level was detected in the nuclei isolated from Brassica uninucleate microspores as well as in mature Triticum pollen. Interestingly, diploid nuclei were detected in both binucleate and mature pollen of B. napus. The possible origins of the diploid nuclei are discussed.

Activation of platelets in whole blood by recombinant factor VIIa by a thrombin-dependent mechanism.

Using a diluted whole blood method of flow cytometric analysis, we have shown that platelets could be activated in vitro in the presence of high concentrations (100 nmol/l) of recombinant factor (F) VIIa (rFVIIa; NovoSeven(R)) and 2.5 mmol/l calcium chloride. This was demonstrated by a significant increase in the mean percentage of platelets expressing CD62P and their mean fluorescent intensity (MFI) after 30 min versus platelets incubated with calcium or rFVIIa alone or diluted blood alone. The presence of rFVIIa and calcium increased the exposure of the PAC-1 activation epitope of glycoprotein (Gp) IIb/IIIa. This effect was equally influenced by the presence of calcium alone but not by rFVIIa. The effect of rFVIIa was time and concentration dependent. Thrombin generation was also necessary, as the effect of rFVIIa was completely abrogated by the additional presence of hirudin. Furthermore, soy bean trypsin inhibitor (SBTI) but not corn trypsin inhibitor (CTI) abrogated CD62P exposure, suggesting that thrombin was derived via FX but not FXII activation. Exposure of CD62P demonstrated a significant lag phase, sometimes of the order of > 30 min, as well as large intersubject variation. Significant platelet activation was observed at a concentration as low as 25 nmol/l rFVIIa. Platelet-leucocyte aggregation was also increased in the presence of 25 nmol/l rFVIIa and calcium. No significant difference was observed between levels of CD62P in diluted whole blood and platelet-rich plasma adjusted to an identical platelet count after their exposure to rFVIIa and calcium for 30 min.

Androgen receptor expression in archival human breast tumors.

Hormone receptors play a major role in growth and hormonal therapy of breast and prostate tumors. Quantitative results from the ligand binding assays cannot determine heterogeneity in receptor expression nor can they discriminate between expression of the stromal and the tumor cells. Availability of antibodies to hormone receptors has led to the development of immunohistochemistry as a standard method for monitoring of hormone receptor expression under a microscope. However, this method is based on examination of a small number of cells. Laser flow cytometry has been extensively used for monitoring of receptor expression in human liquid tumors. As most of the hormone receptor expression is nuclear, we have developed methods for flow cytometric analysis of receptor expression in nuclei isolated from enzyme treated paraffin sections. The present report based on gated analysis of androgen receptor expression in nuclei isolated from archival formalin fixed/paraffin embedded breast tumors shows that receptor expression in aneuploid sub-populations is greater than that of the diploid cells.