Determinants of platelet conjugate formation with polymorphonuclear leukocytes or monocytes in whole blood

2007 ◽  
Vol 98 (12) ◽  
pp. 1276-1284 ◽  
Author(s):  
Simona Costanzo ◽  
Branislav Vohnout ◽  
Licia Iacoviello ◽  
Giovanni de Gaetano ◽  
Benedetta Izzi ◽  
...  
Keyword(s):  
Platelet Count ◽  
Whole Blood ◽  
Polymorphonuclear Leukocytes ◽  
Large Population ◽  
Mixed Cell ◽  
Cd11b Expression ◽  
Glucose Levels ◽  
Primary Platelet ◽  
Collagen Stimulation

SummaryFollowing preliminary in-vitro experiments, platelet-leukocyte conjugates and their determinants were evaluated in citrated whole blood from 349 subjects (209 women, age 16–92 years) randomly recruited from the general population. Platelet activation by ADP/collagen but not leukocyte stimulation by fMLP or LTB4 resulted in formation of platelet conjugates with PMN or monocytes. In the population study, mixed cell conjugates, platelet P-selectin and leukocyte CD11b were measured by flow cytometry both at baseline and after in-vitro stimulation with ADP/collagen. The latter significantly increased platelet conjugates with either PMN or monocytes, platelet P-selectin and leukocyte CD11b expression. Platelet count significantly correlated with platelet-PMN, platelet-monocyte conjugates and P-selectin both at baseline and upon stimulation. In all conditions, both conjugate levels correlated with each other, when adjusted for gender, age and platelet count. Age correlated with platelet-PMN conjugate numbers in basal and stimulated conditions and with basal P-selectin. ADP/collagen stimulation resulted in higher P-selectin and conjugates values in women. Among risk factors, a significant correlation was found between conjugate and glucose levels. In conclusion, the presence and formation in whole blood from a large population of plateletleukocyte conjugates reflects primary platelet – but not leukocyte – activation and varies with gender, age, platelet count and blood glucose.

A Simple and Rapid Method to Determine GPIIb/IIIa-Receptor Inhibitor Activity in Whole Blood

1999 ◽  
Vol 19 (03) ◽  
pp. 134-138
Author(s):  
Gitta Kühnel ◽  
A. C. Matzdorff
Keyword(s):  
Flow Cytometry ◽  
Platelet Count ◽  
Blood Sample ◽  
Platelet Activation ◽  
Whole Blood ◽  
Receptor Occupancy ◽  
Aggregate Formation ◽  
Inhibitor Activity ◽  
Receptor Inhibitor

SummaryWe studied the effect of GPIIb/IIIa-inhibitors on platelet activation with flow cytometry in vitro. Citrated whole blood was incubated with increasing concentrations of three different GPIIb/IIIa-inhibitors (c7E3, DMP728, XJ757), then thrombin or ADP were added and after 1 min the sample was fixed. Samples without c7E3 but with 0.1 U/ml thrombin had a decrease in platelet count. Samples with increasing concentrations of c7E3 had a lesser or no decrease in platelet count. The two other inhibitors (DMP 725, XJ757) gave similar results. GPIIb/IIIa-inhibitors prevent aggregate formation and more single platelets remain in the blood sample. The agonist-induced decrease in platelet count correlates closely with the concentration of the GPIIb/IIIa inhibitor and receptor occupancy. This correlation may be used as a simple measure for inhibitor activity in whole blood.

Early Platelet-Collagen Interactions in Whole Blood and Their Modifications by Aspirin and Dipyridamole Evaluated by a New Method (Basic Wave)

1985 ◽  
Vol 54 (04) ◽  
pp. 799-803 ◽  
Author(s):  
José Luís Pérez-Requejo ◽  
Justo Aznar ◽  
M Teresa Santos ◽  
Juana Vallés
Keyword(s):  
Whole Blood ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
White Blood Cells ◽  
Reversible Aggregation ◽  
Inhibitory Compounds ◽  
Rapid Generation ◽  
Stimulation Of ◽  
Collagen Stimulation

SummaryIt is shown that the supernatant of unstirred whole blood at 37° C, stimulated by 1 μg/ml of collagen for 10 sec, produces a rapid generation of pro and antiaggregatory compounds with a final proaggregatory activity which can be detected for more than 60 min on a platelet rich plasma (PRP) by turbidometric aggregometry. A reversible aggregation wave that we have called BASIC wave (for Blood Aggregation Stimulatory and Inhibitory Compounds) is recorded. The collagen stimulation of unstirred PRP produces a similar but smaller BASIC wave. BASIC’s intensity increases if erythrocytes are added to PRP but decreases if white blood cells are added instead. Aspirin abolishes “ex vivo” the ability of whole blood and PRP to generate BASIC waves and dipyridamole “in vitro” significantly reduces BASIC’s intensity in whole blood in every tested sample, but shows little effect in PRP.

Abstract 571: A Novel High-Throughput Whole Blood Immuno-Counting Assay is Useful in the Assessment of Platelet Responses to Antiplatelet Therapy

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Marie Lordkipanidze ◽  
Natalia Dovlatova ◽  
Mohammad Algahtani ◽  
Martina H Lundberg ◽  
Timothy D Warner ◽  
...  
Keyword(s):  
Platelet Count ◽  
Antiplatelet Therapy ◽  
Platelet Function ◽  
Whole Blood ◽  
Dual Antiplatelet Therapy ◽  
Whole Blood Assay ◽  
Blood Assay ◽  
Treated Sample ◽  
Function Testing

Background: The current gold standard in platelet function testing, light transmission aggregometry, is time- and labor-intensive, and uses platelet-rich plasma which makes it sub-optimal for high throughput testing. In order to reduce blood manipulation prior to platelet function testing and to study multiple platelet activation pathways simultaneously, we have developed a 96-well plate-based assay carried out in whole blood, where aggregation is measured as a decrease in the number of fluorescently-labeled single platelets by flow cytometry. Aim: To investigate whether a 96-well plate-based whole blood assay can be used to assess platelet function. Methods: Platelet function in response to 5 concentrations of lyophilized arachidonic acid (AA), ADP, collagen, epinephrine, TRAP, U46619, and ristocetin, was assessed in healthy volunteers (n=20) to establish normal ranges. The effect of antiplatelet drugs was assessed in vitro by incubation with aspirin (100 μM), cangrelor (1 μM) or both (n=20), and in patients on dual antiplatelet therapy (n=20). After addition of 40 μl of whole blood per well, the plate was shaken for 5 min at 1000 rpm at 37°C; a fixative solution (Platelet Solutions, Nottingham) was applied to stop platelet aggregation and allow analysis in a central laboratory. Fixed whole blood samples (stable for up to 9 days) were labeled with FITC-conjugated CD42a and assessed by flow cytometry. Aggregation was calculated as (Platelet count in vehicle-treated sample - Platelet count in agonist-stimulated sample) / Platelet count in vehicle-treated sample x 100. Results: Dose-response curves were readily assessable for all agonists and intra-individual variability was minimal in healthy volunteers (CV<10%). In vitro addition of aspirin alone resulted in inhibition of AA- and collagen-induced aggregation, whereas cangrelor induced a shift in dose-response to most agonists in addition to profound inhibition of ADP responses. In patients on dual antiplatelet therapy, the pattern of response was consistent with the results obtained with in vitro agents. Conclusions: A 96-well plate-based whole blood assay with a minimal blood volume requirement (<2 ml) could be used to provide a global portrait of platelet responses to antiplatelet agents.

The hepoxilin stable analogue, PBT-3, inhibits primary, platelet-related hemostasis in whole blood measured in vitro with the PFA-100

2003 ◽  
Vol 112 (4) ◽  
pp. 245-248 ◽  
Author(s):  
Denis Reynaud ◽  
Dewi Clark ◽  
Na Qiao ◽  
Margaret L Rand ◽  
Cecil R Pace-Asciak
Keyword(s):  
Whole Blood ◽  
Primary Platelet

scholarly journals Validation of Blood Collection Procedures for the Determination of Circulating Vascular Endothelial Growth Factor (VEGF) in Different Blood Compartments

2001 ◽  
Vol 16 (2) ◽  
pp. 87-96 ◽  
Author(s):  
R. Dittadi ◽  
S. Meo ◽  
F. Fabris ◽  
G. Gasparini ◽  
D. Contri ◽  
...  
Keyword(s):  
Platelet Count ◽  
Whole Blood ◽  
Relevant Information ◽  
Sampling Procedure ◽  
Systematic Evaluation ◽  
Blood Collection ◽  
Sample Collection ◽  
Analytical Phase

Aims of the study. Studies on circulating VEGF have reported mixed results, possibly due to a lack of standardization of the pre-analytical phase. The aim of our investigation was to standardize the sampling procedure for the determination of VEGF in different blood fractions. Basic procedures. We evaluated various clotting times for obtaining serum in 30 subjects, as well as different procedures for the preparation of plasma Edinburgh anticoagulant mixture (EDTA, PGE1, theophylline) and CTAD. VEGF was also assayed in lysed whole blood. In vitro platelet activation was monitored by measuring the levels of PF4. VEGF and PF4 were measured using commercially available enzyme-linked immunoassays. Main findings. Clotting time increased the release of VEGF, which reached a plateau between 2 and 4 hours. The percent increase of VEGF at 2 hours ranged from 118% to 4515% (median 327%) compared to samples centrifuged within 10 min from withdrawal. VEGF was not different and PF4 was very low or undetectable in Edinburgh plasma and CTAD plasma, while it was significantly higher in sodium citrate plasma. VEGF in CTAD plasma was not correlated with platelet count or leukocytes. Serum VEGF did not correlate with the leukocyte number, but it correlated significantly with the platelet count. Principal conclusions. The procedures for sample collection described above are highly standardized and easy to perform in a routine setting. We therefore suggest systematic evaluation of VEGF in CTAD plasma, in serum (clotting for 2 hours at room temperature) and in whole blood, until prospective controlled clinical studies will have clarified in which blood compartment(s) VEGF provides clinically relevant information.

scholarly journals Efficacy of a Semi Automated Commercial Closed System for Autologous Leukocyte- and Platelet-Rich Plasma (l-prp) Production in Dogs: A Preliminary Study

Animals ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 1342 ◽  
Author(s):  
Roberta Perego ◽  
Eva Spada ◽  
Luciana Baggiani ◽  
Piera Anna Martino ◽  
Daniela Proverbio
Keyword(s):  
Platelet Count ◽  
Closed System ◽  
Whole Blood ◽  
Platelet Rich Plasma ◽  
In Vivo Studies ◽  
High Concentration ◽  
Commercial System ◽  
Thrombin Activation

Background: To characterize the cellular composition (platelets, erythrocytes, and leukocytes) and determine platelet-derived growth factor isoform BB (PDGF-BB) concentration in canine leukocyte- and platelet rich plasma (L-PRP) produced using a commercial semi-automated closed system. Methods: Twenty milliliters of citrated whole blood were obtained from 30 healthy un-sedated canine blood donors and processed using a semi-automated completely closed commercial system (CPUNT 20, Eltek group, Casale Monferrato, Alessandria, Italy) according to the manufacturer’s instructions. Erythrocyte, leukocyte, and platelet counts were determined in both whole blood (WB) and resultant L-PRP. The PDGF-BB concentration was evaluated after bovine thrombin activation of 10 L-PRP samples. Results: This commercial system produced on average 2.3 ± 0.7 mL of L-PRP containing a high concentration of platelets (767,633 ± 291,001 μL, p < 0.001), with a 4.4 fold increase in platelet count, lower concentration of erythrocytes (528,600 ± 222,773 μL, p < 0.001) and similar concentration of leukocytes (8422 ± 6346 μL, p = 0.9918) compared with WB. L-PRP had an average of 3442 ± 2061 pg/mL of PDGF-BB after thrombin activation. Neutrophils, lymphocytes and monocytes average percent content in L-PRP was 14.8 ± 13.2, 71.7 ± 18.5 and 10.7 ± 6.4, respectively. Conclusion: Sterile canine L-PRP prepared using this semi-automated closed system is easy to obtain, produces a significant increase in platelet count compared to WB and contains a detectable concentration of PDGF-BB after activation. Additional in vitro and in vivo studies are needed to assess inflammatory markers concentration and the therapeutic efficacy of this L-PRP in dogs.

Thirty-minutes’ exposure to smartphone call triggers neutrophil activation in vitro

2016 ◽  
Vol 54 (9) ◽  
pp. 1497-1501 ◽  
Author(s):  
Giuseppe Lippi ◽  
Elisa Danese ◽  
Giorgio Brocco ◽  
Marco Benati ◽  
Gian Luca Salvagno ◽  
...  
Keyword(s):  
Whole Blood ◽  
Polymorphonuclear Leukocytes ◽  
Complete Blood Count ◽  
Close Contact ◽  
Activation Parameters ◽  
Median Percentage ◽  
Blood Samples ◽  
Leukocyte Counts ◽  
Potential Impact

Abstract Background: Despite accumulating evidence about the negative health effects of exposure to electromagnetic fields emitted by mobile phones, no information is available on the potential impact of radiofrequency (RF) waves on polymorphonuclear leukocytes biology. Methods: Two sequential whole blood tubes were collected from 16 ostensibly healthy volunteers. After placing the former tube of each subject in a plastic rack, 1 cm from a commercial smartphone (carrier frequency, 900 MHz), a call was placed on the smartphone and a communication lasting 30 min was manually activated. The latter blood tube of each volunteer was placed in another plastic rack, for an identical period of time, avoiding close contact with sources of RF waves. A complete blood count was then assessed in all whole blood samples, using Advia 2120. Results: The 30-min exposure of blood to RF waves did not induce significant variations of total and differential leukocyte counts. A significant decrease was however observed for many neutrophils parameters, with median percentage variation of −3.9% for the lobularity index (LI), −29.8% for the myeloperoxidase index (MPXI), −0.6% for the neutrophil cluster mean x (NEUTx) and −0.7% for the neutrophil cluster mean y (NEUTy), respectively. The percentage of blood samples with reduced values after exposure to RF waves was 81% for LI, 88% for NEUTx and 100% for both MPXI and NEUTy. Conclusions: The results of this study show that exposure to smartphone RF waves triggers activation of neutrophils in vitro, as mirrored by the significant variations observed in many activation parameters in Advia 2120.

Polymorphonuclear leukocytes L-selectin expression decreases as they age in circulation

1997 ◽  
Vol 272 (1) ◽  
pp. H401-H408 ◽  
Author(s):  
S. F. Van Eeden ◽  
S. Bicknell ◽  
B. A. Walker ◽  
J. C. Hogg
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Life Span ◽  
Whole Blood ◽  
Half Life ◽  
Polymorphonuclear Leukocytes ◽  
In Vitro Studies ◽  
Double Immunolabeling

We recently reported that L-selectin expression increases on circulating polymorphonuclear leukocytes (PMN) during active bone marrow release, which suggests that older cells in the circulation have lower levels of L-selectin than those recently released from the bone marrow. The present study was designed to test the hypothesis that L-selectin expression reduces on PMN as they age in the circulation. In vitro studies using flow cytometry showed that PMN L-selectin decreased to 14.6 +/- 2.3% of baseline during a 24-h incubation at 37 degrees C. To test this hypothesis in vivo, rabbit PMN, labeled in vivo with 5'-bromo-2-deoxyuridine (PMN-brdU) in donor animals, were infused into recipient rabbits as whole blood (n = 5) and followed over 24 h in the circulation with a double immunolabeling technique. These results showed that the fraction of the L-selectin-negative PMN-BrdU in the circulation increased with time (P < 0.001), and nearly all of the PMNBrdU in the circulation were L-selectin negative after 24 h. Removal of L-selectin from the surface of PMN-BrdU with chymotrypsin before infusion did not change their rate of removal from the circulation (half-life or t 1/2 262 vs. 296 min, P = NS). We conclude that there is a continuous loss of L-selectin from PMN during their life span in the circulation, which supports the hypothesis that L-selectin expression decreases on PMN as they age.

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