scholarly journals Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

10.3791/53823 ◽  
2016 ◽  
Author(s):  
Britt Van Aelst ◽  
Hendrik B. Feys ◽  
Rosalie Devloo ◽  
Philippe Vandekerckhove ◽  
Veerle Compernolle
Keyword(s):  
Platelet Transfusion ◽  
Microfluidic Flow

In vitro thromboelastometric evaluation of the efficacy of frozen platelet transfusion

2015 ◽  
Vol 136 (2) ◽  
pp. 348-353 ◽  
Author(s):  
Antonio Pérez-Ferrer ◽  
Ricardo Navarro-Suay ◽  
Aurora Viejo-Llorente ◽  
Maria J. Alcaide-Martín ◽  
Jesús de Vicente-Sánchez ◽  
...  
Keyword(s):  
Platelet Transfusion

scholarly journals Adult, but Not Neonatal, Platelet Transfusions Drive a Monocyte Trafficking Phenotype in Vitro and In Vivo

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2144-2144
Author(s):  
Preeti Maurya ◽  
Sara Ture ◽  
Kathleen E. McGrath ◽  
James Palis ◽  
Craig N. Morrell
Keyword(s):  
Platelet Transfusion ◽  
Age Groups ◽  
In Vivo Model ◽  
Mouse Bone Marrow ◽  
Monocyte Migration ◽  
Ccr2 Expression ◽  
Increased Risk ◽  
Platelet Transfusions

Abstract Although, thrombocytopenia can affect all age groups, neonates, especially pre-term, have an increased incidence of thrombocytopenia. Platelet transfusions may reduce the bleeding risk in neonates, but are also associated with adverse short and long-term immune and inflammatory outcomes. A randomized trial of platelet transfusions in neonates found that transfusion was associated with an increased risk of necrotizing enterocolitis, unilateral/bilateral retinopathy, and bronchopulmonary dysplasia. Past work from our research team found that neonatal platelets expressed lower levels of mRNA for many immune related molecules compared to adult platelets. We therefore sought to determine whether the transfusion of adult platelets to neonates resulted in developmental immune dysregulation, with a focus on platelet and monocyte interactions. To explore the interactions between monocytes and platelets, we isolated monocytes from adult mouse bone marrow and co-incubated monocytes with adult (>8 weeks old) or neonatal mouse platelets (7 days old mice) and determined inflammatory and trafficking monocyte phenotypes by flow cytometry and qRT-PCR. Monocytes treated with adult platelets had an increased inflammatory (Ly6C hi) and trafficking phenotype (CCR2 hi), while monocytes treated with neonatal platelets adopted an inflammatory, but not trafficking phenotype. As expected, adult platelets increased the expression of monocyte inflammatory (Nos2, Cxcl1, Ccl2) and trafficking (Ccr2) mRNA, while neonatal platelets also increased inflammatory mRNA expression, but did not increase Ccr2 expression. Adult platelets express more Selp (P-selectin) than neonatal platelets and P-selectin is a major mediator of platelet and monocyte interactions. We confirmed that adult platelets expressed more P-selectin protein compared to neonatal platelets, and found that blocking P-selectin decreased adult platelet induced CCR2 expression to levels similar to monocytes treated with neonatal platelets. Using a transwell chamber we assessed adult and neonatal platelet effects on monocyte migration towards the CCR2 ligand CCL2. Monocytes were treated with adult platelets had significantly greater monocyte migration compared to monocytes co-incubated with neonatal platelets. To model platelet transfusions in the setting of thrombocytopenia, we used 14d old thrombopoietin receptor knockout mice (TPOR -/-) that have low platelet counts, and infused adult or neonatal platelets. We observed a significant increase in inflammatory and trafficking monocytes in mice transfused with adult platelets compared to those transfused with neonatal platelets. Using an in vivo model of monocyte chemotaxis, mice were treated with CCL2 intraperitoneal after platelet transfusion. Adult platelet transfusions, but not neonatal, increased monocyte peritoneal trafficking to CCL2. These data provide comparative insights as to how adult and neonatal platelet transfusions regulate monocyte functions. Adult platelet transfusions to neonates are associated with an inflammatory and trafficking monocyte phenotype that is platelet P-selectin dependent and may have a major impact on neonatal platelet transfusion complications. Disclosures Palis: Rubius Therapeutics: Consultancy.

PLATELET TRANSFUSION THERAPY - FUTURE TRENDS AND FEASIBLE GOALS

1987 ◽  
Author(s):  
C Th Smit Sibinga
Keyword(s):  
Platelet Transfusion ◽  
Storage Conditions ◽  
Future Trends ◽  
Transfusion Therapy ◽  
Life Saving ◽  
Immunological Effects ◽  
Alternative Approaches ◽  
And Storage ◽  
The Devil

It is better to know than not to know the devil. Platelet transfusion therapy is one such devil. While the therapy initially is a benefit and might even be life-saving, it often implicates future problems which prohibit optimal efficacy.The factors determining both the beneficial and the prohibitive effects are complex and not seldom masked. Problem areas include the logistics of availability, the prevention of immunological effects and incompatibilities, the prevention of trans- missable diseases and the prediction of clinical efficacy.Efforts for optimising platelet transfusion therapy should focus on safety, purity, potency and clinical efficacy. However, each of these premises has to be outlined with precision in order to allow targetted development.Major areas of attention will be the selection and utilisation of available resources, the options for purification, efforts to optimise processing, preservation and storage conditions, innovation of the in vitro viability concept and alternative approaches to selection in order to increase the predictive value of compatibility testing.

Recurrence of Severe ITP Following Administration of G-CSF: Remission Following Platelet and Vinca-Alkaloid Infusion.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3222-3222
Author(s):  
Eugene R. Ahn ◽  
John J. Byrnes ◽  
Vincenzo Fontana ◽  
Pamela Dudkiewicz ◽  
Carlos J. Bidot ◽  
...  
Keyword(s):  
Platelet Count ◽  
Platelet Transfusion ◽  
Ex Vivo ◽  
Vinca Alkaloid ◽  
High Dose ◽  
Vinca Alkaloids ◽  
Platelet Counts ◽  
Life Threatening ◽  
Immune Neutropenia

Abstract Introduction: In ITP, platelets opsonized with antibodies are phagocytosed by macrophages. Activation of macrophages often triggers aggravation or relapses of ITP as demonstrated following vaccination, infections. G-CSF stimulates granulocyte colonies but can stimulate macrophages at higher concentrations in vitro. We report recurrence of severe life threatening ITP following G-CSF therapy, successfully managed by selective injury of macrophages with sequential infusions of platelets and vinca-alkaloids. Case Study: A 30 year old healthy Caucasian man developed severe ITP in 9/03 with wet purpura, epistaxis, multiple hematomas in the mouth, tongue and lips and a platelet count <2 K. He suffered severe headaches, refractory gastrointestinal (GI) and genitourinary (GU) bleeding requiring numerous platelet and pRBC transfusions. Increased megakaryocytes were seen in a bone marrow biopsy. CT scans of the head and body were normal, including normal spleen size. ITP was refractory to several measures including high dose glucocorticoids, IV immunoglobulins (IVIG), danazol, rituximab, and vinca-alkaloids. Splenectomy in 5/04 induced a complete remission, lasting for over 3 years. On 2/12/07 he presented with agranulocytosis and neutropenic fever. His Hgb and platelet counts were normal but leukocyte count was 0.9 with absent granulocytes. IVIG infusions began for immune neutropenia with partial improvement of granulocytopenia. Beginning 5/31/07, he was treated with a biweekly regimen of IVIG and Neulasta with normalization of WBC. However, a month following this normalization, patient presented with a platelet count of 9K, wet purpura, epistaxis, multiple hematomas in the tongue and oral mucosa, GI and GU bleeding, headaches and dizzy spells. In spite of high dose IV steroids, daily platelet and pRBC transfusions were required, with little change in platelet counts. He also suffered hypotensive episodes from GI bleeding and pseudomonas bacteremia. Using a rationale described in our previous work (NEJM298:1101, 1978), vincristine 1mg injection was given immediately following platelet transfusion and one week later, 4mg vinblastine immediately following another platelet transfusion. Vinca rapidly binds to transfused platelets and serve as targeted therapy against the activated macrophages that phagocytose platelets. The therapy was effective. Platelet count rose to 72K 1 week after vinblastine, and then normalized. Additional vincristine 1mg was given at discharge. ITP underwent remission. Summary/Discussion: A patient with refractory ITP who underwent CR for over three years after splenectomy suffered severe life threatening thrombocytopenia following injections of G-CSF. This case report is highlighted by the following features. While ITP was in CR, severe granulocytopenia developed which responded to IVIG, indicating an autoimmune cause of leukopenia. Treatment with G-CSF for leukopenia triggered recurrence of severe ITP. Platelet transfusion immediately followed by injection of vinca-alkaloids was successful in inducing remission of life threatening ITP. G-CSF should be used with caution in patients with history of ITP, since it may activate macrophages and trigger relapse of ITP. The immediate sequence of platelet transfusion followed by vinca injection might be particularly useful in this scenario, and is less cumbersome compared to the previously described procedure of incubating platelets ex-vivo with vinca prior to infusion (NEJM298:1101, 1978; AmJHem81:423, 2006).

Ex Vivo Expanded Megakaryocyte Progenitors Generated From Human Mobilized Peripheral Blood CD34+ Stem Cells Can Be Cryopreserved and Transplanted Into NSG Mice to Generate Functional Platelets

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 482-482
Author(s):  
Holger Karsunky ◽  
Robert J. Tressler ◽  
Joy Chananukul
Keyword(s):  
Stem Cells ◽  
Peripheral Blood ◽  
Platelet Transfusion ◽  
Ex Vivo ◽  
High Dose ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Mobilized Peripheral Blood ◽  
Megakaryocyte Progenitors

Abstract Abstract 482 Thrombocytopenia is a common side effect of high-dose chemotherapy that can compromise cancer treatment by requiring treatment delay and/or dose reduction for the patient. Platelet transfusion is typically given to prevent severe hemorrhage. However, several factors including acquisition, banking, and associated risks of bacterial infections and alloimmunization are hampering reliance on platelet transfusion. Growth factors are also used to stimulate proliferation and differentiation of megakaryocytes to increase platelet production, but in severely myelosuppressed patients these have only had modest benefit. The limitations of these two modalities for the treatment of chemotherapy-induced thrombocytopenia indicates that additional treatment approaches are needed. We have developed a novel approach to reconstitute megakaryocytes and platelets in thrombocytopenic patients which is presented here. We have identified a scalable culture system using serum-free medium and a defined cytokine cocktail free of animal products to expand CD34+ hematopoietic stem cells from G-CSF mobilized peripheral blood donors in vitro and direct their development to the megakaryocyte lineage to yield committed human megakaryocyte progenitors (MKPs). These MKPs can be readily cryopreserved while retaining their capacity to generate CFU-MK and platelets in vitro. When infused into NSG mice, ex vivo expanded MKP generate clinically relevant platelet levels of platelets in blood within a few days with sustained platelet levels for several weeks. The platelets generated from MKP in vivo are also functional as assessed by CD62P expression in responses to ADP stimulation in vitro. Our results present a compelling approach for the development of off-the-shelf storable MKPs for the treatment of thrombocytopenia. Disclosures: Karsunky: Cellerant Therapeutics Inc.: Employment, Patents & Royalties. Tressler:Cellerant Therapeutics, Inc.: Employment, Equity Ownership. Chananukul:Cellerant Therapeutics Inc.: Employment, Patents & Royalties.

scholarly journals Studies on Swelling Behavior of HPMC based Matrix Tablets by the Application of Digital Imaging Technique

1970 ◽  
Vol 7 (2) ◽  
pp. 169-175
Author(s):  
Irwin Dewan ◽  
Md Elias-Al-Mamun ◽  
Reza-ul Jalil
Keyword(s):  
Digital Photography ◽  
Biological Tissues ◽  
Matrix Tablets ◽  
Widespread Application ◽  
Microfluidic Flow ◽  
Hydrogel Matrix ◽  
Hydrophobic Materials ◽  
The Matrix ◽  
Swelling Rate

The widespread application of hydrogels in a number of applications like control of microfluidic flow, development of muscle-like actuators, filtration/separation and drug delivery makes it important to properly understand these materials. Understanding hydrogel properties is also important from the standpoint of their similarity to many biological tissues. The degree of swelling as well as the rate of swelling of the hydrogel was studied through experiments using digital photography. First of all the matrix was prepared by blending properly weighed HPMC, Theophylline and excipients in a laboratory mixture for 10 minutes followed by compression with 5 ton force for 30 seconds. The study was designed to evaluate the effect of hydrophobic materials on HPMC matrix tablets in terms of in vitro swelling study. Different viscosity grades of HPMC i.e. 6- cps, 15-cps and 50-cps were used to prepare tablets along with various hydrophobic materials. Among the hydrophobic materials stearic acid (SA), cetyl alcohol (CA), bees wax (BW), glyceryl monostearate (GMS), cetostearyl alcohol (CSA) and stearyl alcohol (STA) were used. It was found that presence of hydrophobic ingredients in the HPMC hydrogel matrix substantially reduces swelling of the matrix. In this work, the extent of swelling of HPMC matrix was evaluated by using digital photography and counting the pixels with Photoshop software. Finally, it was shown from the experiment that the rate and extent of drug release is mainly governed by swelling rate of different HPMC viscosity grade used and the nature of hydrophobic materials included into the matrix system. Key words: Swelling, Matrix, HPMC, Hydrogel, Photography. doi: 10.3329/dujps.v7i2.2174 Dhaka Univ. J. Pharm. Sci. 7(2): 169-175, 2008 (December)

scholarly journals Pseudothrombocytopenia Inducing Nonindicated Platelet Transfusion after Cardiac Surgery

2021 ◽  
Vol 2021 ◽  
pp. 1-4
Author(s):  
Enise Ceran ◽  
Christine Schlömmer ◽  
Ivonne Kröckel ◽  
Georg Scheriau ◽  
Philipp Angleitner ◽  
...  
Keyword(s):  
Cardiac Surgery ◽  
Diagnostic Tests ◽  
Platelet Transfusion ◽  
Ethylenediaminetetraacetic Acid ◽  
Cell Counting ◽  
Inadequate Response ◽  
Automated Cell Counting ◽  
Low Platelet Count ◽  
Caucasian Female

Pseudothrombocytopenia (PTCP) is an in vitro phenomenon of low platelet count caused by the agglutination of platelets, leading to false low platelet counts in automated cell counting. Typically, ethylenediaminetetraacetic acid (EDTA) mediates this platelet clumping. PTCP has little clinical significance, but misdiagnosis may lead to unnecessary diagnostic tests and treatment. In this case report, we present a 65-year-old Caucasian female suffering from multiple complications during and after cardiac surgery. During her postoperative stay at the ICU, she was diagnosed with thrombocytopenia and an inadequate response to platelet supplementation.

A Small Molecule Inhibitor of the Gas6/Mer Pathway Inhibits Platelet Activation and Thrombosis with Equal Efficacy to, but Greater Potency Than, iMer, the Novel MerTK Splice Variant

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3303-3303
Author(s):  
Brian R. Branchford ◽  
Luke Law ◽  
Susan Sather ◽  
Gary Brodsky ◽  
H. Shelton Earp ◽  
...  
Keyword(s):  
Surface Area ◽  
Platelet Activation ◽  
Artery Occlusion ◽  
Area Coverage ◽  
Average Percentage ◽  
Microfluidic Flow ◽  
Molecule Inhibitor ◽  
Vehicle Treated Control

Abstract Abstract 3303 Background: Growth Arrest Specific gene 6 (Gas6) signals through platelet-surface Mer receptors, leading to platelet activation and thrombus stabilization via activation of PI3K and Akt, and β3 integrin phosphorylation. This amplifies outside-in signaling via αIIb β3, a necessary step for stable platelet aggregation. iMer is a truncated form of the Mer receptor tyrosine kinase's extracellular domain, produced by alternative splicing, that inhibits Gas6 signaling. A selective UNC Mer small molecule inhibitor (UNC Mer TKI) inhibits signaling by inhibiting Mer tyrosine phosphorylation. Objectives: We hypothesized that inhibiting the Gas6/Mer pathway with UNC Mer TKI would decrease platelet activation responses and thrombus formation to a greater degree than iMer. So, we comparatively evaluated iMer's and UNC Mer TKI's inhibition of Gas6/Mer signaling in vitro and in vivo. Methods: We measured the effect on platelet inhibiton of iMer and UNC Mer TKI using lab assays of human platelet function and murine thrombosis models. In vitro studies included standard aggregometry and aggregate formation on collagen surfaces in a microfluidic flow chamber. In vivo studies included a FeCl3-induced model of carotid artery injury and a collagen/epinephrine-induced pulmonary embolism (PE) model to compare thrombosis protection between littermate C57BL/6 mice treated with inhibitors or vehicle control. A paired t-test was used to compare samples in aggregation, microfluidic flow surface area coverage, as well as elapsed time to initial and stable occlusions in the FeCl3model and survival time in the PE model. Results/Discussion: Inhibitor-treated platelets exhibited significantly decreased aggregation. Collagen-stimulated samples treated with 1.2 μM iMer had mean aggregation of 45 +/− 13% (Stand Dev), compared to 72 +/− 8% in controls (n=6, p=0.02). 1.2 μM UNC Mer TKI-treated samples had mean aggregation of 56 +/−15%, compared to 74 +/−10% in controls (n=5, p<0.01). Adhesion of platelets to collagen under physiologic flow conditions resulted in 1.9% (+/− 1%) coverage in 1.2 μM iMer-treated samples compared to 8.7 +/− 2% mean surface area coverage in controls (n=7, p<0.001). 1.2 μM UNC Mer TKI-treated samples exhibited 8.5 +/−8.8% surface area coverage, compared to 19.2 +/− 15.7% for control-treated samples (n=5 in quadruplicate, p<0.01), and 2.4 +/−1.7% for abciximab-treated positive controls. Both iMer- and UNC Mer TKI-treated samples have a higher average percentage of small platelet aggregates (1–10 platelets/aggregate) compared to controls, which had a higher average percentage of large aggregates (>10 platelets/aggregate). Following 6% FeCl3-induced carotid artery injury for analysis of iMer's thrombosis protection, vehicle-treated control mice (n=6) had a hazard ratio for stable artery occlusion of 7.9 (95%CI 1.7–36.3) compared to 30mg/kg iMer-treated mice (n=8, p<0.01). The 1.2 μM iMer treated mice also demonstrated significantly prolonged time to both initial (p=0.03) and stable occlusion (p=0.02), suggesting decreased thrombus stability. In analysis of UNC Mer TKI's thrombosis inhibition, vehicle-treated control mice (n=10) had a hazard ratio for stable artery occlusion of 4.8 (95%CI 1.4–15.8) compared 3 mg/kg UNC Mer TKI-treated mice (n=9, p=0.02). Times to initial (p=0.06) and stable (p<0.01) occlusions were also prolonged in the UNC Mer TKI-treated mice compared to controls. In the PE model, 1.2 μM iMer-treated mice (n=5) had longer mean survival (14.1 +/− 14.5 min) following collagen/epinephrine injection than controls (n=5, 1.78 +/12.4 min, p=NS), with 2 iMer–treated mice and no controls surviving for 30 min. A much greater effect was seen with the 3 mg/kg UNC Mer TKI-treated mice (n=5) who had a mean survival time of 37.24 +/−31.2 min, with 3 surviving for a full 60 min, compared to 1.63 +/− 0.6 min (p=0.03) for controls (n=5), of which only 1 survived for 60 min. Conclusions: iMer and UNC Mer TKI are Gas6/Mer inhibitors that decrease platelet activation and protect mice from thrombosis, and may have translational applications as novel anti-platelet agents. UNC Mer TKI has similar efficacy, and higher potency, especially in the animal models where similar (FeCl3 model) or superior (PE model) results were seen with a 10-fold lower concentration. UNC Mer TKI also has the advantage of possible oral preparation, which may increase its potential therapeutic appeal. Disclosures: Branchford: University of Colorado: This author has provisional patent considerations for iMer, This author has provisional patent considerations for iMer Patents & Royalties. Sather:University of Colorado: This author has provisional patent considerations for iMer, This author has provisional patent considerations for iMer Patents & Royalties. Brodsky:University of Colorado: This author has provisional patent considerations for iMer, This author has provisional patent considerations for iMer Patents & Royalties. Graham:University of Colorado: This author has provisional patent considerations for iMer, This author has provisional patent considerations for iMer Patents & Royalties.

scholarly journals Towards Reduction or Substitution of Cytotoxic DMSO in Biobanking of Functional Bioengineered Megakaryocytes

2020 ◽  
Vol 21 (20) ◽  
pp. 7654
Author(s):  
Denys Pogozhykh ◽  
Dorothee Eicke ◽  
Oleksandr Gryshkov ◽  
Willem F. Wolkers ◽  
Kai Schulze ◽  
...  
Keyword(s):  
Platelet Transfusion ◽  
Ex Vivo ◽  
Practical Implementation ◽  
Stock Management ◽  
Promising Alternative ◽  
Efficient Treatment ◽  
Before And After ◽  
Cryopreservation Techniques

Donor platelet transfusion is currently the only efficient treatment of life-threatening thrombocytopenia, but it is highly challenged by immunological, quality, and contamination issues, as well as short shelf life of the donor material. Ex vivo produced megakaryocytes and platelets represent a promising alternative strategy to the conventional platelet transfusion. However, practical implementation of such strategy demands availability of reliable biobanking techniques, which would permit eliminating continuous cell culture maintenance, ensure time for quality testing, enable stock management and logistics, as well as availability in a ready-to-use manner. At the same time, protocols applying DMSO-based cryopreservation media were associated with increased risks of adverse long-term side effects after patient use. Here, we show the possibility to develop cryopreservation techniques for iPSC-derived megakaryocytes under defined xeno-free conditions with significant reduction or complete elimination of DMSO. Comprehensive phenotypic and functional in vitro characterization of megakaryocytes has been performed before and after cryopreservation. Megakaryocytes cryopreserved DMSO-free, or using low DMSO concentrations, showed the capability to produce platelets in vivo after transfusion in a mouse model. These findings propose biobanking approaches essential for development of megakaryocyte-based replacement and regenerative therapies.

Action of HL-A Specific Antibodies on Platelets in Vitro

1975 ◽  
Author(s):  
D. Heinrich ◽  
C. Kessler ◽  
U. Stephinger ◽  
W. Kunkel ◽  
C. Mueller-Eckhardt
Keyword(s):  
Platelet Transfusion ◽  
Complement Fixation ◽  
Serotonin Release ◽  
Serotonin Uptake ◽  
Clot Retraction ◽  
Specific Antibodies ◽  
Specific Antisera ◽  
Platelet Spreading

Refractoriness to platelet transfusion in thrombocytopenic patients often is due to HL-A isoimmunisation. HL-A antibody-induced platelet alteration in vivo is associated with a reduction of platelet survival.Action of HL-A specific antibodies on platelets in vitro was studied applying serological (absorption/elution-experiments; micro-complement fixation), morphological (platelet spreading, electron microscopy) and functional parameters (clot retraction, platelet agglutination, 14C-serotonin uptake and 14C-serotonin release).Results: Incubation of platelets (PRP) with HL-A specific antisera induced a specific platelet agglutination with degranulation and 14C-serotonin release, furthermore a specific inhibition of 14C-serotonin uptake and clot retraction. Experiments with Prostaglandin E15 Apyrase, Heparin, ASA and EDTA in connection with a HL-A specific LDH-liberation from platelets suggest a lytic, complement-dependent action of HL-A antibodies on platelets in plasma (PRP).Experiments with washed platelets and HL-A specific eluates (D. Heinrich et al.: Vox Sang. 27, 310, 1974) indicate a second, nonlytic action of HL-A antibodies on platelets. Cofactors of the latter reaction are Ca++, fibrinogen and thrombin.

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