Only the Truth Would Enlighten Us — The Advantages and Disadvantages of Flow Cytometry as a Method of Choice in the Study of Mouse and Rat Platelets

SummaryP-selectin, also known as CD62P, GMP140 or PADGEM, is present in platelet a-granules and endothelial cell Weibel-Palade bodies and is very rapidly expressed on the surface of these cells on activation. In this study, an anti P-selectin monoclonal antibody (LYP20) was used, in tandem with flow cytometry, to identify activated platelets at the site of induced vascular trauma or in peripheral blood. Moreover, electron microscopy was performed to characterize sites of vascular trauma and quantify the number of adhering platelets. The same induced vascular trauma was observed to result into animals responding in 2 different ways (Group I, Group II) following the degree of platelet activation. Five rats, out of 14 with induced vascular trauma, had more than half of their circulating platelets expressing P-selectin when drawn at the site of the trauma (67.4% ± 3.44) or in peripheral blood (78.5% ± 2.5) (Group I). In the remaining 9 animals a much smaller proportion of circulating platelets expressed P-selectin when assayed from trauma sites (18% ± 3.34) or in peripheral blood (18.0% ± 4.30) (Group II). Enhanced P-selectin expression by circulating platelets in Group I, compared to Group II, appears to be linked to the degree of activated platelets adhering at sites of trauma (171 ± 15 × 103 platelets versus 48 ± 31 × 103 platelets per mm2). In the 5 control animals, that were not operated on, platelets expressing P-selectin when drawn at the site of a mock trauma (7.0% ± 1.84) or in the peripheral blood (11.2% ± 3.30) showed little activation. In addition, no platelet adhesion was seen on the vascular bed of these animals. Results from this study show that analysis of P-selectin (CD62P) expression, in circulating platelets, is a valuable and rapid marker of platelet activation following severe vascular trauma induced in rats. However, activated platelets were not detected to the same extent in the peripheral blood of all animals having undergone vascular trauma. It is conceivable that platelets, depending on the degree of activation, may be actively sequestered in organs and prevented from circulating. Alternatively, P-selectin may be rapidly endocytosed, or not expressed, by activated circulating platelets depending on the type of agonists implicated in vivo activation.

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Flow Cytometry and Real-Time Quantitative PCR as Tools for Assessing Plasmid Persistence

Applied and Environmental Microbiology ◽

10.1128/aem.00793-14 ◽

2014 ◽

Vol 80 (17) ◽

pp. 5439-5446 ◽

Cited By ~ 11

Author(s):

Wesley Loftie-Eaton ◽

Allison Tucker ◽

Ann Norton ◽

Eva M. Top

Keyword(s):

Flow Cytometry ◽

Real Time ◽

Quantitative Pcr ◽

High Throughput ◽

Performance Criteria ◽

Plate Count ◽

Real Time Quantitative Pcr ◽

Advantages And Disadvantages ◽

Free Cells ◽

Loss Rates

ABSTRACTThe maintenance of a plasmid in the absence of selection for plasmid-borne genes is not guaranteed. However, plasmid persistence can evolve under selective conditions. Studying the molecular mechanisms behind the evolution of plasmid persistence is key to understanding how plasmids are maintained under nonselective conditions. Given the current crisis of rapid antibiotic resistance spread by multidrug resistance plasmids, this insight is of high medical relevance. The conventional method for monitoring plasmid persistence (i.e., the fraction of plasmid-containing cells in a population over time) is based on cultivation and involves differentiating colonies of plasmid-containing and plasmid-free cells on agar plates. However, this technique is time-consuming and does not easily lend itself to high-throughput applications. Here, we present flow cytometry (FCM) and real-time quantitative PCR (qPCR) as alternative tools for monitoring plasmid persistence. For this, we measured the persistence of a model plasmid, pB10::gfp, in threePseudomonashosts and in known mixtures of plasmid-containing and -free cells. We also compared three performance criteria: dynamic range, resolution, and variance. Although not without exceptions, both techniques generated estimates of overall plasmid loss rates that were rather similar to those generated by the conventional plate count (PC) method. They also were able to resolve differences in loss rates between artificial plasmid persistence assays. Finally, we briefly discuss the advantages and disadvantages for each technique and conclude that, overall, both FCM and real-time qPCR are suitable alternatives to cultivation-based methods for routine measurement of plasmid persistence, thereby opening avenues for high-throughput analyses.

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In vitro assays for cell death determination

Archive of oncology ◽

10.2298/aoo0804049j ◽

2008 ◽

Vol 16 (3-4) ◽

pp. 49-54 ◽

Cited By ~ 15

Author(s):

Vladimir Jurisic ◽

Vladimir Bumbasirevic

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

Western Blot ◽

In Vitro Assays ◽

Activity Determination ◽

Advantages And Disadvantages ◽

Enzymes Activity ◽

Death Determination

In this paper, we focused on commonly used in vitro assays for estimation of cell death: morphological analyses of cell death, cytotoxic assays based on enzymes activity determination, flow cytometry, and western blot techniques. We discussed advantages and disadvantages of several assays used in the modern research for estimation of cell death.

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Flow Cytometry in the Detection of Neonatal Sepsis

International Journal of Pediatrics ◽

10.1155/2013/763191 ◽

2013 ◽

Vol 2013 ◽

pp. 1-6 ◽

Cited By ~ 18

Author(s):

Volker N. Umlauf ◽

Stephan Dreschers ◽

Thorsten W. Orlikowsky

Keyword(s):

Flow Cytometry ◽

Antimicrobial Therapy ◽

Neonatal Sepsis ◽

Clinical Symptoms ◽

Advantages And Disadvantages ◽

Laboratory Markers ◽

Hla Dr ◽

Initial Symptoms ◽

Diagnostic Pitfalls

Neonatal sepsis remains a burden problem by showing minimal initial symptoms of subtle character, nonspecific manifestation, and diagnostic pitfalls. The clinical course can be fulminant and fatal if treatment is not commenced promptly. It is therefore crucial to establish early diagnosis and initiate adequate therapy. Besides clinical symptoms, the most reliable laboratory markers in establishing diagnosis is currently the combined measurement of CRP and a cytokine (IL-6 and IL-8). Due to their different kinetics, a diagnostic gap might occur and thus withholding antimicrobial therapy in clinical suspicion of infection is not acceptable. We therefore need parameters which unerringly differentiate between infants in need for antimicrobial therapy and those who are not. Flow cytometry promises to be a useful tool in this field, allowing the determination of different cellular, dissolved, and functional pathophysiological components of sepsis. Despite technical and methodical advances in flow cytometry, its use in clinical routine is still limited. Advantages and disadvantages of promising new parameters in diagnosis of sepsis performed by flow cytometry, particularly CD64, HLA-DR, and apoptosis, are reviewed here. The necessity of tests to be used as an “ideal” parameter is presented.

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Technical Aspects of Minimal Residual Disease Detection by Multicolor Flow Cytometry in Acute Myeloid Leukemia Patients

Clinical oncohematology ◽

10.21320/2500-2139-2021-14-4-503-512 ◽

2021 ◽

Vol 14 (4) ◽

pp. 503-512

Author(s):

I.V. Galtseva ◽

Yuliya Olegovna Davydova ◽

N.M. Kapranov ◽

K.A. Nikiforova ◽

E.N. Parovichnikova

Keyword(s):

Flow Cytometry ◽

Acute Myeloid Leukemia ◽

Minimal Residual Disease ◽

Myeloid Leukemia ◽

Residual Disease ◽

Multicolor Flow Cytometry ◽

Advantages And Disadvantages ◽

Essential Components ◽

Minimal Residual ◽

Acute Myeloid

Detection and monitoring of minimal residual disease (MRD) are essential components of programmed therapy. They are crucial for the choice of treatment strategy and for prognostic purposes practically in all hematologic diseases. MRD is often detected by multicolor flow cytometry, the method with fairly high specificity and sensitivity. However, to identify MRD in acute myeloid leukemia patients is one of the most challenging tasks flow cytometry specialists are faced with. Cytometric data analysis requires the expert knowledge of immunophenotype of all maturing bone marrow cells. Besides, MRD analysis in acute myeloid leukemia has not been standardized while approaches suggested by different studies vary considerably. The present paper reports the experience of MRD analysis, demonstrates the gating strategy, immunophenotype description of normal non-tumor hematopoietic cells, and presents some examples of MRD assessment. Additionally, panels of monoclonal antibodies are provided, along with an evaluation of their advantages and disadvantages.

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Minimal residual disease diagnostics in acute lymphoblastic leukemia: need for sensitive, fast, and standardized technologies

Blood ◽

10.1182/blood-2015-03-580027 ◽

2015 ◽

Vol 125 (26) ◽

pp. 3996-4009 ◽

Cited By ~ 237

Author(s):

Jacques J. M. van Dongen ◽

Vincent H. J. van der Velden ◽

Monika Brüggemann ◽

Alberto Orfao

Keyword(s):

Flow Cytometry ◽

Acute Lymphoblastic Leukemia ◽

Minimal Residual Disease ◽

High Throughput ◽

High Throughput Sequencing ◽

Residual Disease ◽

Lymphoblastic Leukemia ◽

Pcr Analysis ◽

Advantages And Disadvantages ◽

Minimal Residual

Abstract Monitoring of minimal residual disease (MRD) has become routine clinical practice in frontline treatment of virtually all childhood acute lymphoblastic leukemia (ALL) and in many adult ALL patients. MRD diagnostics has proven to be the strongest prognostic factor, allowing for risk group assignment into different treatment arms, ranging from significant treatment reduction to mild or strong intensification. Also in relapsed ALL patients and patients undergoing stem cell transplantation, MRD diagnostics is guiding treatment decisions. This is also why the efficacy of innovative drugs, such as antibodies and small molecules, are currently being evaluated with MRD diagnostics within clinical trials. In fact, MRD measurements might well be used as a surrogate end point, thereby significantly shortening the follow-up. The MRD techniques need to be sensitive (≤10−4), broadly applicable, accurate, reliable, fast, and affordable. Thus far, flow cytometry and polymerase chain reaction (PCR) analysis of rearranged immunoglobulin and T-cell receptor genes (allele-specific oligonucleotide [ASO]-PCR) are claimed to meet these criteria, but classical flow cytometry does not reach a solid 10−4, whereas classical ASO-PCR is time-consuming and labor intensive. Therefore, 2 high-throughput technologies are being explored, ie, high-throughput sequencing and next-generation (multidimensional) flow cytometry, both evaluating millions of sequences or cells, respectively. Each of them has specific advantages and disadvantages.

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Flow Cytometry and Polymerase Chain Reaction-Based Analyses of Minimal Residual Disease in Chronic Lymphocytic Leukemia

Advances in Hematology ◽

10.1155/2010/272517 ◽

2010 ◽

Vol 2010 ◽

pp. 1-11 ◽

Cited By ~ 4

Author(s):

Sabrina Uhrmacher ◽

Felix Erdfelder ◽

Karl-Anton Kreuzer

Keyword(s):

Flow Cytometry ◽

Chronic Lymphocytic Leukemia ◽

Minimal Residual Disease ◽

Residual Disease ◽

Lymphocytic Leukemia ◽

Daily Routine ◽

Color Flow ◽

Advantages And Disadvantages ◽

Igh Rearrangement ◽

Minimal Residual

New therapeutic strategies developed recently for chronic lymphocytic leukemia (CLL) have led to remarkable treatment response rates and complete hematological remissions. This means highly sensitive and specific techniques are increasingly needed to evaluate minimal residual disease (MRD) in CLL patients. Quantitative MRD levels can be used as prognostic markers, where total MRD eradication is associated with prolonged survival. Nowadays, PCR and flow cytometry techniques used to detect MRD in CLL patients can generate reliable and quantitative results with the highest sensitivity. MRD Flow is based on four-color flow cytometry using specific antibody combinations. For allele specific oligonucleotide real-time quantification (ASO RQ) PCR individual primers are designed to detect a specific immunoglobulin heavy chain (IgH) rearrangement in each patient clone. Five comprehensive studies investigated and compared the sensitivity and specificity of both methods. Groups of patients receiving different therapies were analyzed at different time points to generate quantitative MRD levels and MRD kinetics. All studies confirmed that both methods generate equivalent results with regard to sensitivity and MRD quantification, although each method has advantages and disadvantages in the daily routine of a standard hematological laboratory. Here, we review these investigations and compare their results in the light of modern therapies.

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Cryogenic atomic force microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100084570 ◽

1991 ◽

Vol 49 ◽

pp. 54-55

Author(s):

K. A. Fisher ◽

M. G. L. Gustafsson ◽

M. B. Shattuck ◽

J. Clarke

Keyword(s):

Room Temperature ◽

Rapid Freezing ◽

Cryogenic Temperatures ◽

Soft Or ◽

Electrically Conductive ◽

Force Microscopy ◽

Flexible Molecules ◽

Advantages And Disadvantages ◽

Atomic Force ◽

Chemical Perturbation

The atomic force microscope (AFM) is capable of imaging electrically conductive and non-conductive surfaces at atomic resolution. When used to image biological samples, however, lateral resolution is often limited to nanometer levels, due primarily to AFM tip/sample interactions. Several approaches to immobilize and stabilize soft or flexible molecules for AFM have been examined, notably, tethering coating, and freezing. Although each approach has its advantages and disadvantages, rapid freezing techniques have the special advantage of avoiding chemical perturbation, and minimizing physical disruption of the sample. Scanning with an AFM at cryogenic temperatures has the potential to image frozen biomolecules at high resolution. We have constructed a force microscope capable of operating immersed in liquid n-pentane and have tested its performance at room temperature with carbon and metal-coated samples, and at 143° K with uncoated ferritin and purple membrane (PM).

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A conduction-cooled stage for high-resolution Cryo-SEM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100131048 ◽

1992 ◽

Vol 50 (2) ◽

pp. 1282-1283

Author(s):

John G. Sheehan

Keyword(s):

High Resolution ◽

Liquid Nitrogen ◽

Mechanical Stability ◽

Cooling System ◽

Cold Trap ◽

Nitrogen Gas ◽

Particulate Suspensions ◽

Advantages And Disadvantages ◽

Convection Cooling ◽

Styrene Butadiene

The goal is to examine with high resolution cryo-SEM aqueous particulate suspensions used in coatings for printable paper. A metal-coating chamber for cryo-preparation of such suspensions was described previously. Here, a new conduction-cooling system for the stage and cold-trap in an SEM specimen chamber is described. Its advantages and disadvantages are compared to a convection-cooling system made by Hexland (model CT1000A) and its mechanical stability is demonstrated by examining a sample of styrene-butadiene latex.In recent high resolution cryo-SEM, some stages are cooled by conduction, others by convection. In the latter, heat is convected from the specimen stage by cold nitrogen gas from a liquid-nitrogen cooled evaporative heat exchanger. The advantage is the fast cooling: the Hexland CT1000A cools the stage from ambient temperature to 88 K in about 20 min. However it consumes huge amounts of liquid-nitrogen and nitrogen gas: about 1 ℓ/h of liquid-nitrogen and 400 gm/h of nitrogen gas. Its liquid-nitrogen vessel must be re-filled at least every 40 min.

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Computer simulations for the TEM bend contour technique

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100163137 ◽

1996 ◽

Vol 54 ◽

pp. 134-135

Author(s):

Vladimir Yu. Kolosov ◽

Anders R. Thölén

Keyword(s):

Computer Simulations ◽

Real Space ◽

Advantages And Disadvantages ◽

Short Overview ◽

Diffraction Contrast ◽

Beam Method ◽

Convergent Beam ◽

Contour Technique

In this paper we give a short overview of two TEM applications utilizing the extinction bend contour technique (BC) giving the advantages and disadvantages; especially we consider two areas in which the BC technique remains unique. Special attention is given to an approach including computer simulations of TEM micrographs.BC patterns are often observed in TEM studies but are rarely exploited in a serious way. However, this type of diffraction contrast was one of the first to be used for analysis of imperfections in crystalline foils, but since then only some groups have utilized the BC technique. The most extensive studies were performed by Steeds, Eades and colleagues. They were the first to demonstrate the unique possibilities of the BC method and named it real space crystallography, which developed later into the somewhat similar but more powerful convergent beam method. Maybe, due to the difficulties in analysis, BCs have seldom been used in TEM, and then mainly to visualize different imperfections and transformations.

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