scholarly journals Brief Note: The Use of a Special Preservation Medium for the Maintenance of Platelet Viability at 4 C

Blood ◽  
1960 ◽  
Vol 15 (6) ◽  
pp. 909-917 ◽  
Author(s):  
MARIO BALDINI ◽  
SHIRLEY EBBE ◽  
WILLIAM DAMESHEK ◽  
Janet Donovan
Keyword(s):  
Phosphate Buffer ◽  
Platelet Survival ◽  
Rabbit Platelets ◽  
Preservation Medium

Abstract Viability of rabbit platelets stored for 1 to 6 days in different media at 4 C. was studied using the Cr51-platelet survival technic. Platelets stored for 24 hours in Triton-saline or DAS-gelatin had practically no viability. When platelets were stored for 24 hours in diluted plasma, significant degrees of viability were found, but this decreased rapidly in the successive 48 hours. The viability of platelets stored in diluted plasma containing inosine, adenine, glucose and phosphate buffer was maintained at significant levels over a six day period. The data presented here suggest that viability and, consequently, the hemostatic properties of platelets stored at 4 C. can be maintained over relatively long periods of time provided that loss of the ATP reserve of the cells can be prevented.

THE EFFECT OF ENZYMATIC TREATMENT OF RABBIT PLATELETS ON THE BINDING OF IMMUNOGLOBULIN G TO THE SURFACE

1987 ◽  
Author(s):  
R M Evans ◽  
M A Packham
Keyword(s):  
Immunoglobulin G ◽  
Rabbit Serum ◽  
Membrane Glycoproteins ◽  
Plasmin Activity ◽  
Platelet Suspension ◽  
Platelet Survival ◽  
Igg Binding ◽  
Rabbit Igg ◽  
Rabbit Platelets

As shown previously, cleavage of surface glycoproteins on the platelet membrane shortens platelet survival. Since immunoglobulins have been implicated in the condition of idiopathic thrombocytopenic purpura in which patients have an elevated level of platelet-associated immunoglobulin G (I g G) and shortened platelet survival, we measured the binding of IgG to rabbit platelets that had been treated with neuraminidase or plasmin, to determine whether increased IgG binding to these platelets might be the signal that leads to their accelerated clearance. Rabbit platelets were washed and resuspended in calcium-free Tyrode-albumin solution (1.0 x 106/ μL), pH 6.5, and then incubated with either neuraminidase (0.1 U/mL at 22°C) or plasmin (activity: 1.6 ymol BAEE/min/mL platelet suspension at 37¼C) for 30 min. The platelets were then centrifuged and resuspended in heat-inactivated rabbit serum for 30 min to allow IgG to bind to the surface. After additional washing steps to remove non-specifically bound IgG, the amount Qt IgG that associated with the platelets was measured using an 125I-labeled goat anti-rabbit IgG binding assay. Binding data were analyzed by plotting the calculated fg of IgG bound/platelet versus the amount of 125-1 goat anti-rabbit IgG added. The binding curves for both neuraminidase and plasmin indicated significantly increased amounts of specifically bound IgG compared to control platelets. At a concentration of 30 μg/mL of labeled goat antirabbit IgG added, the amount of label associated with untreated rabbit platelets had plateaued at 0.36± 0.11 fg/platelet whereas the amount associated with neuraminidase-treated platelets was 5 times greater and with pi asmin-treated platelets, 1.5 times greater, and neither had plateaued. The results obtained with plasmin are of particular interest since this proteolytic enzyme shortens platelet survival and is present in vivo during thromboembolic events. These results indicate that IgG binds specifically to the surface of rabbit platelets following enzymatic alterations of the membrane glycoproteins and therefore may represent at least one mechanism that determines which platelets are cleared by the reticuloendothelial system.

Platelet sialic acid and platelet survival after aggregation by ADP

Blood ◽  
1980 ◽  
Vol 56 (5) ◽  
pp. 876-880
Author(s):  
MA Packham ◽  
MA Guccione ◽  
RL Kinlough-Rathbone ◽  
JF Mustard
Keyword(s):  
Sialic Acid ◽  
Human Platelets ◽  
Platelet Surface ◽  
Release Reaction ◽  
Dense Granules ◽  
Total Sialic Acid ◽  
Platelet Survival ◽  
Rabbit Platelets ◽  
Induced Aggregation

Some investigators have reported recently that platelet surface sialic acid is decreased during ADP-induced aggregation, whereas others have reported an increase. Since removal of sialic acid from the platelet surface shortens platelet survival, we have determined the survival of platelets that have been aggregatad by ADP. We have also measured the amount of sialic acid in the suspending fluid of platelets after ADP- induced aggregation. ADP-induced aggregation did not cause the loss of sialic acid from rabbit platelets (which do not undergo a release reaction in response to ADP) nor from washed human platelets in a medium containing physiologic concentrations of calcium in which granule contents are not released. In a medium without added calcium, ADP caused the release of 14C-serotonin (42.5% +/- 3%) from human platelets, but less than 4% of the sialic-acid-containing material was released. It seems likely that little of the releasable sialic acid of platelets is in the dense granules or the alpha-granules. Thrombin (5 U/ml) released 90.0% +/- 3.4% of the serotonin from human platelets but only 20.6% +/- 7.4% of the total sialic-acid-containing material. Neuraminidase removed 42.3% of the total sialic acid, presumably from the platelet surface. Rabbit platelets that had been aggregated by ADP and deaggregated survived normally when returned to the circulation. This observation also provides evidence that they had not lost membrane sialic acid during aggregation and deaggregation.

scholarly journals Platelet sialic acid and platelet survival after aggregation by ADP

Blood ◽  
1980 ◽  
Vol 56 (5) ◽  
pp. 876-880 ◽  
Author(s):  
MA Packham ◽  
MA Guccione ◽  
RL Kinlough-Rathbone ◽  
JF Mustard
Keyword(s):  
Sialic Acid ◽  
Human Platelets ◽  
Platelet Surface ◽  
Release Reaction ◽  
Dense Granules ◽  
Total Sialic Acid ◽  
Platelet Survival ◽  
Rabbit Platelets ◽  
Induced Aggregation

Abstract Some investigators have reported recently that platelet surface sialic acid is decreased during ADP-induced aggregation, whereas others have reported an increase. Since removal of sialic acid from the platelet surface shortens platelet survival, we have determined the survival of platelets that have been aggregatad by ADP. We have also measured the amount of sialic acid in the suspending fluid of platelets after ADP- induced aggregation. ADP-induced aggregation did not cause the loss of sialic acid from rabbit platelets (which do not undergo a release reaction in response to ADP) nor from washed human platelets in a medium containing physiologic concentrations of calcium in which granule contents are not released. In a medium without added calcium, ADP caused the release of 14C-serotonin (42.5% +/- 3%) from human platelets, but less than 4% of the sialic-acid-containing material was released. It seems likely that little of the releasable sialic acid of platelets is in the dense granules or the alpha-granules. Thrombin (5 U/ml) released 90.0% +/- 3.4% of the serotonin from human platelets but only 20.6% +/- 7.4% of the total sialic-acid-containing material. Neuraminidase removed 42.3% of the total sialic acid, presumably from the platelet surface. Rabbit platelets that had been aggregated by ADP and deaggregated survived normally when returned to the circulation. This observation also provides evidence that they had not lost membrane sialic acid during aggregation and deaggregation.

Modifications of Platelets that Affect Platelet Adherence to Collagen or Damaged Aorta aud Platelet Survival

1975 ◽  
Author(s):  
J.-P. Cazenave ◽  
H.-J. Reimers ◽  
J. Greenberg ◽  
S. Niewiarowski ◽  
M. A. Packham ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Survival Time ◽  
Platelet Adhesion ◽  
Sodium Periodate ◽  
Platelet Adherence ◽  
Platelet Survival ◽  
No Release ◽  
Rabbit Platelets ◽  
Glass Surfaces

Platelet changes which govern their removal from the circulation are poorly understood We have modified platelets by thrombin degranulation, treatment with plasmin, removing surface sialic acid with neuraminidase, or exposure to sodium periodate. Washed rabbit platelets were prelabeled with 51Cr so that their adherence to collagen-coated glass surfaces or to intima of damaged aortas could be quantitated and their survival in vivo studied (Cazenave et al., J. Lab. Clin. Med. 82, 978, 1973; Reimers et al., Br. J. Haemat, 25, 675, 1973). The thrombin-treated platelets from which more than 70% of the serotonin had been released, showed decreased adherence to collagen (by 43±5%) and decreased adherence to damaged aorta (by 74 ±2%). Survival time for thrombin-treated platelets was 82±2 hr and for control platelets, 82±2 hr. Platelets treated with plasmin were insensitive to ADP unless fibrinogen were added. Plasmin treatment diminished platelet adherence to collagen by 40±5% and to damaged aorta by 68±2%. The survival time was 93±2 hr for plasmin-treated platelets (controls, 88±3 hr). Treatment with purified neuraminidase removed 8-35% of total platelet sialic acid with no release of granule contents. This did not inhibit platelet adherence to collagen or damaged aorta. If more than 15% of the sialic acid were removed, the platelets were cleared from the circulation within 1 hr. Exposure of platelets to NaIO4 (0.1-1 mM) for up to 10 min diminished adherence to collagen by 47 ±4%. When platelets were treated with 0.5 to 1 mM NaIO4 more than 70% of them were cleared from the circulation within 1 hr. Thus, treatments which alter platelet interaction with surfaces do not necessarily affect platelet survival, whereas removal of surface sialic acid shortens platelet- survival but does not influence platelet adhesion to surfaces. NaIO4 affects both adherence and survival but it may affect several platelet components.

Investigation of In Vivo Factors that May Influence Platelet Survival and Density

1979 ◽  
Author(s):  
M.A. Packham ◽  
J.F. Mustard ◽  
M.A. Guccione ◽  
P. D. Winocour ◽  
H.M. Groves ◽  
...  
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Platelet Membrane ◽  
Membrane Glycoproteins ◽  
Aortic Endothelium ◽  
Platelet Survival ◽  
Rabbit Platelets

Platelet survival CPS) is shortened in a number of conditions but the mechanisms responsible are unclear. In rabbits, removal of the aortic endothelium or injury of the neointima does not shorten PS. However, induction of thrombi in rabbit aortae with Indwelling cannulae (IDC) shortens FS (IDC 37.0 hr, control 79.6 hr), and Increases the proportion of platelets in the lightest fraction upon straetan density gradient centrifugation. Therefore we examined the effect of agents to which platelets may be exposed during thromboembolism (ADP, thrombin, plasmin) on PS and platelet density. ADP treatnent of washed rabbit platelets did not alter their survival but did increase the proportion in the lightest fraction. Treatment of platelets with thrombin did not shorten PS but increased the proportion in the lightest fraction. Treatment with plasmin in vitro shortened PS (plasmin, 57.6 ± 6.0 hr, control 80.2 ± 4,2 hr) and increased the proportion in the lightest fraction. Thus changes in platelet density are not necessarily associated with changes in PS. Of the factors investigated that are known to be involved in thromboembolism, only plasmin shortened PS. This may be due to its ability to alter major platelet membrane glycoproteins(principally glycoproteins I and II of rabbit platelets).

Freeze-fracture, freeze-etch complementary pairs of virus-infected cells reveal value of etching even when relatively high concentrations of cryoprotectants are used

1978 ◽  
Vol 36 (2) ◽  
pp. 136-137
Author(s):  
Russell L. Steere ◽  
Eric F. Erbe
Keyword(s):  
Plant Tissue ◽  
Phosphate Buffer ◽  
Infected Plant ◽  
Freeze Fracture ◽  
Distilled Water ◽  
Virus Infected Plant ◽  
Infected Cells ◽  
High Concentrations

It has been assumed by many involved in freeze-etch or freeze-fracture studies that it would be useless to etch specimens which were cryoprotected by more than 15% glycerol. We presumed that the amount of cryoprotective material exposed at the surface would serve as a contaminating layer and prevent the visualization of fine details. Recent unexpected freeze-etch results indicated that it would be useful to compare complementary replicas in which one-half of the frozen-fractured specimen would be shadowed and replicated immediately after fracturing whereas the complement would be etched at -98°C for 1 to 10 minutes before being shadowed and replicated.Standard complementary replica holders (Steere, 1973) with hinges removed were used for this study. Specimens consisting of unfixed virus-infected plant tissue infiltrated with 0.05 M phosphate buffer or distilled water were used without cryoprotectant. Some were permitted to settle through gradients to the desired concentrations of different cryoprotectants.

Spontaneous Cataract in Nude Athymic Mice

1977 ◽  
Vol 35 ◽  
pp. 512-513
Author(s):  
Ronald H. Bradley ◽  
R. S. Berk ◽  
L. D. Hazlett
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Nude Mouse ◽  
Cellular Immunity ◽  
Phosphate Buffer ◽  
Osmium Tetroxide ◽  
Athymic Mice ◽  
Transmission Microscopy ◽  
Mouse Lens ◽  
Scanning Electron

The nude mouse is a hairless mutant (homozygous for the mutation nude, nu/nu), which is born lacking a thymus and possesses a severe defect in cellular immunity. Spontaneous unilateral cataractous lesions were noted (during ocular examination using a stereomicroscope at 40X) in 14 of a series of 60 animals (20%). This transmission and scanning microscopic study characterizes the morphology of this cataract and contrasts these data with normal nude mouse lens.All animals were sacrificed by an ether overdose. Eyes were enucleated and immersed in a mixed fixative (1% osmium tetroxide and 6% glutaraldehyde in Sorenson's phosphate buffer pH 7.4 at 0-4°C) for 3 hours, dehydrated in graded ethanols and embedded in Epon-Araldite for transmission microscopy. Specimens for scanning electron microscopy were fixed similarly, dehydrated in graded ethanols, then to graded changes of Freon 113 and ethanol to 100% Freon 113 and critically point dried in a Bomar critical point dryer using Freon 13 as the transition fluid.

Visualization of Antibody Transport in Rat Visceral Yolk-Sac Placenta

1982 ◽  
Vol 40 ◽  
pp. 246-247
Author(s):  
William P. Jollie
Keyword(s):  
Phosphate Buffer ◽  
Yolk Sac ◽  
Female Rats ◽  
Thin Sections ◽  
Visceral Yolk Sac ◽  
Antibody Complex ◽  
Cytochemical Reaction ◽  
Hind Foot ◽  
Antigen Antibody ◽  
Yolk Sac Placenta

A technique has been developed for visualizing antibody against horseradish peroxidase (HRP) in rat visceral yolk sac, the placental membrane across which passive immunity previously has been shown to be transferred from mother to young just prior to birth. Female rats were immunized by injecting both hind foot pads with 1 mg HRP emulsified in complete Freund's adjuvant. They were given a booster of 0.5mg HRP in 0.1 ml normal saline i.v. after one week, then bred and autopsied at selected stages of pregnancy, viz., 12, 1 7 and 22 days post coitum, receiving a second booster, injected as above, five days before autopsy. Yolk sacs were removed surgically and fixed immediately in 2% paraformaldehye, 1% glutaraldehye in 0.1 M phosphate buffer with 0.01% CaCl2 at pH 7.4, room temperature, for 3 hr, rinsed 3X in 0.1 M phosphate buffer plus 5% sucrose, then exposed to 1 mg HRP in 1 ml 0.1 M phosphate buffer at pH 7.4 for 1 hr. They were refixed in aldehydes, as above, for 1 5 min (to assure binding of antigen-antibody complex). Following buffer washes, the tissues were incubated in 3 mg diaminobenzidine tetrahydrochloride and 0.01% H2O2 in 0.05 M Tris-HCl buffer for 30 min. After brief buffer washes, they were postfixed in 2% OsO4. in phosphate buffer at pH 7.4, 4°C for 2 hr, dehydrated through a graded series of ethanols, and embedded in Durcupan. Thin sections were observed and photographed without contrast-enhancement with heavy metals. Cytochemical reaction product marked the site of HRP (i.e., antigen) which, in turn, was present only where it was bound with anti-HRP antibody.

Ultrastructure of developing visual cortical synapses in the cat superior colliculus

1991 ◽  
Vol 49 ◽  
pp. 156-157
Author(s):  
Caroline A. Miller ◽  
Laura L. Bruce
Keyword(s):  
Superior Colliculus ◽  
Phosphate Buffer ◽  
Receptive Fields ◽  
Visual Cortical Area ◽  
Cortical Synapses ◽  
Visual Cortical ◽  
And Function ◽  
Phosphate Buffered Saline ◽  
The Brain ◽  
Cat Superior Colliculus

The first visual cortical axons arrive in the cat superior colliculus by the time of birth. Adultlike receptive fields develop slowly over several weeks following birth. The developing cortical axons go through a sequence of changes before acquiring their adultlike morphology and function. To determine how these axons interact with neurons in the colliculus, cortico-collicular axons were labeled with biocytin (an anterograde neuronal tracer) and studied with electron microscopy.Deeply anesthetized animals received 200-500 nl injections of biocytin (Sigma; 5% in phosphate buffer) in the lateral suprasylvian visual cortical area. After a 24 hr survival time, the animals were deeply anesthetized and perfused with 0.9% phosphate buffered saline followed by fixation with a solution of 1.25% glutaraldehyde and 1.0% paraformaldehyde in 0.1M phosphate buffer. The brain was sectioned transversely on a vibratome at 50 μm. The tissue was processed immediately to visualize the biocytin.

Resolution of Channels in the Exine by Translocation of Colloidal Iron

1971 ◽  
Vol 29 ◽  
pp. 352-353
Author(s):  
John R. Rowley
Keyword(s):  
Phosphate Buffer ◽  
Pollen Development ◽  
Osmium Tetroxide ◽  
Pollen Grains ◽  
Deionized Water ◽  
Colloidal Iron ◽  
Fixed Material ◽  
Epilobium Angustifolium ◽  
Untreated Material ◽  
Microspore Mitosis

The morphology of the exine of many pollen grains, at the time of flowering, is such that one can suppose that transport of substances through the exine occurred during pollen development. Holes or channels, microscopic to submicroscopic, are described for a large number of grains. An inner part of the exine of Epilobium angustifolium L. and E. montanum L., which may be referred to as the endexine, has irregularly shaped channels early in pollen development although by microspore mitosis there is no indication of such channeling in chemically fixed material. The nucleus in microspores used in the experiment reported here was in prophase of microspore mitosis and the endexine, while lamellated in untreated grains, did not contain irregularly shaped channels. Untreated material from the same part of the inflorescence as iron treated stamens was examined following fixation with 0.1M glutaraldehyde in cacodylate-HCl buffer at pH 6.9 (315 milliosmoles) for 24 hrs, 4% formaldehyde in phosphate buffer at pH 7.2 (1,300 milliosmoles) for 12 hrs, 1% glutaraldehyde mixed with 0.1% osmium tetroxide for 20 min, osmium tetroxide in deionized water for 2 hrs and 1% glutaraldehyde mixed with 4% formaldehyde in 0.1M cacodylate-HCl buffer at pH 6.9 for two hrs.

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