Novel Methodology for the Detection of Cryptosporidium parvum: A Comparison of Cooled Charge Couple Devices (CCD) and Flow Cytometry

1993 ◽  
Vol 27 (3-4) ◽  
pp. 89-92 ◽  
Author(s):  
Andrew Campbell ◽  
Lucy Robertson ◽  
Huw Smith
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Cryptosporidium Parvum ◽  
Propidium Iodide ◽  
Three Dimensional ◽  
Flow Cytometer ◽  
Cryptosporidium Oocysts ◽  
Exact Size

Flow cytometry and CCD were assessed for their usefialness in the detection of oocysts of Cryptosporidium. Oocysts were labelled with FITC-monoclonal antibody and with the nuclear stains 4’6-diamidino-2-phenyl indole (DAPI) and propidium iodide (PI) before analysis by flow cytometer and CCD. Although the flow cytometer tested was able to concentrate particles and place them on a slide for subsequent viewing, readily sorting oocysts from contaminating debris, confirmation by fluoresence microscopy was still essential, even when additional parameters such as the inclusion of DAPI is used. Initial observations from the use of CCD, however, suggested that screening samples for oocysts was a possibility. Three dimensional visualisation of individual oocysts can map precisiely both the detailed morphology and the exact size of oocysts, thereby making confirmation by fluoresence microscopy unneccesary.

scholarly journals Data-Driven Compensation for Flow Cytometry of Solid Tissues

2011 ◽  
Vol 2011 ◽  
pp. 1-11 ◽  
Author(s):  
Nickolaas Maria van Rodijnen ◽  
Math Pieters ◽  
Sjack Hoop ◽  
Marius Nap
Keyword(s):  
Flow Cytometry ◽  
Propidium Iodide ◽  
Dna Content ◽  
Flow Cytometer ◽  
Data Driven ◽  
Color Flow ◽  
Operator Experience ◽  
Solid Tissues

Propidium Iodide is a fluorochrome that is used to measure the DNA content of individual cells, taken from solid tissues, with a flow cytometer. Compensation for spectral cross-over of this fluorochrome still leads to compensation results that are depending on operator experience. We present a data-driven compensation (DDC) algorithm that is designed to automatically compensate combined DNA phenotype flow cytometry acquisitions. The generated compensation values of the DDC algorithm are validated by comparison with manually determined compensation values. The results show that (1) compensation of two-color flow cytometry leads to comparable results using either manual compensation or the DDC method; (2) DDC can calculate sample-specific compensation trace lines; (3) the effects of two different approaches to calculate compensation values can be visualized within one sample. We conclude that the DDC algorithm contributes to the standardization of compensation for spectral cross-over in flow cytometry of solid tissues.

Microwave Inactivation of Cyclospora cayetanensis Sporulation and Viability of Cryptosporidium parvum Oocysts

2006 ◽  
Vol 69 (8) ◽  
pp. 1957-1960 ◽  
Author(s):  
YNES R. ORTEGA ◽  
JYEYIN LIAO
Keyword(s):  
Cryptosporidium Parvum ◽  
Propidium Iodide ◽  
Cryptosporidium Oocyst ◽  
Cooking Time ◽  
Cyclospora Cayetanensis ◽  
The Third ◽  
Infectivity Assay ◽  
Cryptosporidium Oocysts ◽  
Cryptosporidium Parvum Oocysts ◽  
Cooking Times

The efficacy of microwave heating on the viability of Cryptosporidium parvum oocysts and on the sporulation of Cyclospora cayetanensis oocysts for various periods of cooking times (0, 10, 15, 20, 30, and 45 s) at 100% power was determined. Cyclospora oocysts were stored in 2.5% dichromate at 23°C for 2 weeks, and sporulation rates were then determined. The 4′,6-diamidino-2-phenylindole and propidium iodide vital stain and the neonate animal infectivity assay determined Cryptosporidium oocyst viability. Cryptosporidium oocysts could be completely inactivated with as little as 20 s of cooking time, whereas Cyclospora sporulation was observed up to 45 s. Two of the examined microwave ovens were more effective at reducing sporulation and viability than the third one. Because of the variability of temperature achieved by the various ovens, cooking time was not an accurate parameter for parasite inactivation. Cryptosporidium oocysts could be inactivated only when temperatures of 80°C or higher were reached in the microwave ovens.

Flow cytometry using the monoclonal antibody CD10-Pe/Cy5 is a useful tool to identify follicular lymphoma cells

2001 ◽  
Vol 66 (2) ◽  
pp. 100-106 ◽  
Author(s):  
M. Bellido ◽  
E. Rubiol ◽  
J. Ubeda ◽  
O. Lopez ◽  
C. Estivill ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Follicular Lymphoma ◽  
Lymphoma Cells

Effects of a Monoclonal Antibody to Glycoprotein IIb/IIIa (P256) and of Enzymically Derived Fragments of P256 on Human Platelets

1991 ◽  
Vol 65 (04) ◽  
pp. 432-437 ◽  
Author(s):  
A W J Stuttle ◽  
M J Powling ◽  
J M Ritter ◽  
R M Hardisty
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Calcium Ions ◽  
Human Platelet ◽  
Human Platelets ◽  
In Vivo Detection ◽  
Fibrinogen Binding ◽  
Specific Antagonist

SummaryThe anti-platelet monoclonal antibody P256 is currently undergoing development for in vivo detection of thrombus. We have examined the actions of P256 and two fragments on human platelet function. P256, and its divalent fragment, caused aggregation at concentrations of 10−9−3 × 10−8 M. A monovalent fragment of P256 did not cause aggregation at concentrations up to 10−7 M. P256–induced platelet aggregation was dependent upon extracellular calcium ions as assessed by quin2 fluorescence. Indomethacin partially inhibited platelet aggregation and completely inhibited intracellular calcium mobilisation. Apyrase caused partial inhibition of aggregation. Aggregation induced by the divalent fragment was dependent upon fibrinogen and was inhibited by prostacyclin. Aggregation induced by the whole antibody was only partially dependent upon fibrinogen, but was also inhibited by prostacyclin. P256 whole antibody was shown, by flow cytometry, to induce fibrinogen binding to indomethacin treated platelets. Monovalent P256 was shown to be a specific antagonist for aggregation induced by the divalent forms. In–111–labelled monovalent fragment bound to gel-filtered platelets in a saturable and displaceable manner. Monovalent P256 represents a safer form for in vivo applications

A P-Selectin/CD62P Monoclonal Antibody (LYP-20), in Tandem with Flow Cytometry, Detects in vivo Activated Circulating Rat Platelets in Severe Vascular Trauma

1994 ◽  
Vol 72 (05) ◽  
pp. 745-749 ◽  
Author(s):  
Elza Chignier ◽  
Maud Parise ◽  
Lilian McGregor ◽  
Caroline Delabre ◽  
Sylvie Faucompret ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Activation ◽  
Peripheral Blood ◽  
Vascular Trauma ◽  
Activated Platelets ◽  
Group I ◽  
Group Ii ◽  
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.

PAICA: A Method for Characterizing Platelet-Associated Antibodies - Its Application to the Study of Idiopathic Thrombocytopenic Purpura and to the Detection of Platelet-bound c7E3

1996 ◽  
Vol 76 (06) ◽  
pp. 1020-1029 ◽  
Author(s):  
Laurent Macchi ◽  
Gisèle Clofent-Sanchez ◽  
Gérald Marit ◽  
Claude Bihour ◽  
Catherine Durrieu-Jais ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Idiopathic Thrombocytopenic Purpura ◽  
Antithrombotic Therapy ◽  
Membrane Glycoproteins ◽  
Serum Antibodies ◽  
Platelet Destruction ◽  
Thrombocytopenic Purpura ◽  
Capture Assay ◽  
Specific Membrane

SummaryIn idiopathic thrombocytopenic purpura (ITP), autoantibodies reacting with antigens on the platelet membrane bring about accelerated platelet destruction. We now report PAICA (“Platelet-Associated IgG Characterization Assay”), a method for detecting autoantibodies bound to specific membrane glycoproteins in total platelet lysates. This monoclonal antibody (MAb) capture assay takes into account the fact that antibodies on circulating platelets may be translocated to internal pools as well as being on the surface. A total of twenty ITP patients were examined by PAICA, and the results compared with those obtained by measuring (i) serum antibodies bound to paraformaldehyde-fixed control platelets by ELISA, (ii) IgG bound to the surface of the patient’s own platelets by flow cytometry (PSIgG), (iii) total platelet-associated IgG (PAIgG) by ELISA and (iv) serum antibodies reacting with control platelets by MAIPA (“Monoclonal Antibody-specific Immobilization of Platelet Antigens”). Of twelve patients with elevated PAIgG, nine had increased PSIgG yet eleven reacted positively in PAICA. Of these, eight possessed antibodies directed against GP Ilb-IIIa, two against GP Ib-IX and one patient possessed antibodies directed against GP Ilb-IIIa and GP Ia-IIa respectively. Only seven of the patients possessed serum antibodies detectable by MAIPA. PAICA was also able to detect platelet-associated c7E3 (the chimeric form of Fab fragments of the MAb 7E3) following its infusion during antithrombotic therapy, when it proved more sensitive over a seven-day period than a MAIPA assay adapted for assessing surface-bound antibody. We propose that PAICA provides added sensitivity to the detection of platelet-associated antibodies in immune thrombocytopenias or following therapy with humanized MAbs.

Sign in / Sign up

Export Citation Format

Share Document