Importance of L-selectin–dependent leukocyte–leukocyte interactions in human whole blood

Blood ◽  
2000 ◽  
Vol 95 (9) ◽  
pp. 2954-2959 ◽  
Author(s):  
Debra J. Mitchell ◽  
Pauline Li ◽  
Paul H. Reinhardt ◽  
Paul Kubes
Keyword(s):  
Red Blood Cells ◽  
Whole Blood ◽  
Blood Cells ◽  
Rhodamine 6G ◽  
Model System ◽  
Human Whole Blood ◽  
Secondary Interactions ◽  
First Time ◽  
Rolling Leukocytes ◽  
Deficient Mice

The objective of this study was to investigate whether leukocytes could be recruited by rolling leukocytes in a human whole blood model system. In all experiments, either neutrophils, whole blood, or diluted blood was perfused over immobilized E-selectin. With isolated neutrophils (2 × 105/mL), the free-flowing neutrophils were captured by attached neutrophils to form secondary interactions that resulted in lines of rolling leukocytes. These secondary tethers accounted for 50% to 60% of all interactions and were eliminated by an L-selectin antibody, which also eliminated the lines of rolling leukocytes. Perfusion of whole blood or diluted blood revealed no lines of rolling leukocytes. The addition of red blood cells to isolated neutrophils either in a 1000:1 or a 10:1 ratio also inhibited lines of rolling leukocytes. Leukocytes were fluorescently labeled with rhodamine-6G so that leukocyte–leukocyte interactions could be studied in whole blood. A small number of secondary tethers (less than 20%) occurred and could be reduced by more than 80% with an L-selectin antibody. However, the overall impact on leukocyte recruitment was negligible. Similar experiments were performed using murine whole blood or isolated murine leukocytes. In the absence of red blood cells, murine leukocytes also formed lines of rolling leukocytes on E-selectin, and secondary tethers accounted for 50% of total interactions. However, when murine blood (diluted 1:5 with buffer) was perfused over E-selectin, secondary tethers accounted for only 13% of total interactions. These interactions were completely absent when blood was used from L-selectin–deficient mice. These data demonstrate for the first time that the importance of L-selectin–dependent leukocyte–leukocyte interactions is greatly reduced in whole blood and does not enhance overall recruitment of leukocytes in this physiologic milieu.

scholarly journals The Effect of Rigid Red Blood Cells on Platelet Adhesion in Blood Flow and Implications in Sickle Cell Disease

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 986-986
Author(s):  
Alison Leigh Banka ◽  
Mark Shamoun ◽  
Mario Gutierrez ◽  
Tyler Tanski ◽  
Lola Eniola-Adefeso
Keyword(s):  
Blood Flow ◽  
Sickle Cell Disease ◽  
Red Blood Cells ◽  
Whole Blood ◽  
Blood Cells ◽  
Platelet Adhesion ◽  
Model System ◽  
Cell Disease ◽  
The Impact

Introduction: Sickle cell disease (SCD) occurs due to a mutation in the β-subunit of hemoglobin, causing stiffening of red blood cells (RBCs) and leading to RBC sickling and vaso-occlusive crises (VOC) in SCD patients. While sickled RBCs remain a hallmark of SCD, they are prone to lysis and represent a small fraction of the total RBCs present in patients at a given time. The remaining RBCs maintain a normal, discoid shape and are either healthy or stiff due to polymerization of the hemoglobin β-globin subunit. In healthy blood flow, RBCs form a core in the center of the vessel and the remaining cells, platelets and white blood cells (WBCs), marginate towards the endothelium. However, the increased stiffness of RBCs in SCD disrupts this neat segregation of blood cells to different areas of the blood vessel and can contribute to VOC, the root cause of many acute and chronic complications for SCD patients. Despite the presence of normally shaped, stiffened RBCs in SCD patients, the impact of these RBCs on other cell types in blood flow is currently not well understood. Our laboratory previously demonstrated that the presence of artificially rigidified RBCs leads to an expansion of the RBC core and significantly decreases WBC adhesion to an inflamed endothelium in vitro. Here, we examine the impact of stiffened RBCs on platelet adhesion to a damaged endothelium in vitro by first using a model system with artificially rigidified RBCs and second, utilizing SCD patient blood to further support our model and understand platelet-RBC interactions in SCD patients. Methods: In our model system, we artificially rigidified RBCs taken from healthy donors and reconstituted them into whole blood before perfusing the mixture over an activated, damaged endothelium using a parallel plate flow chamber. We quantified platelet adhesion to the endothelium in comparison to healthy, non-rigidified controls using fluorescent microscopy. To determine if our model findings translated to SCD, we recruited a cohort of hemoglobin SS and SC patients during routine visits and similarly perfused their whole blood over the same damaged endothelium and quantified platelet adhesion. Results and conclusions: The inclusion of artificially rigidified RBCs in otherwise healthy subject blood flow significantly increased platelet adhesion to a damaged endothelium with a maximum increase in platelet adhesion of six-fold over a healthy, non-rigid control in our model system. Both RBC rigidity and the percentage of RBCs that were artificially rigidified had a large impact on the increase in platelet adhesion. SCD platelet adhesion to the damaged endothelium model varied from donor to donor based on variables such as treatment method and disease severity. Overall, this work experimentally elucidates the biophysical impact of stiffened RBCs on platelet adhesion using both an artificial model utilizing healthy blood as well as SCD blood, which can help determine the mechanism of action causing VOC in SCD. Disclosures No relevant conflicts of interest to declare.

scholarly journals Transport of Na+ and HCO3- Out of Red Blood Cells Is Simultaneous with a Chloride Shift in Canine and Human Whole Blood Exposed to CO2-Rich Gas.

1993 ◽  
Vol 43 (1) ◽  
pp. 35-49 ◽  
Author(s):  
Senri HIRAKAWA ◽  
Seiichi SHIMABUKURO ◽  
Kiyoji ASANO ◽  
Taro MINAGAWA ◽  
Hisaya IGUCHI ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Whole Blood ◽  
Blood Cells ◽  
Human Whole Blood

Combined Platelet and Erythrocyte Salvage: Evaluation of a New Filtration-based Autotransfusion Device

2021 ◽  
Author(s):  
Alexandre Mansour ◽  
Benoit Decouture ◽  
Mikaël Roussel ◽  
Charles Lefevre ◽  
Lucie Skreko ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Whole Blood ◽  
Blood Cells ◽  
Plasma Proteins ◽  
Complete Blood Count ◽  
Cell Recovery ◽  
Healthy Human ◽  
Platelet Recovery ◽  
Human Whole Blood ◽  
And Function

Background The SAME device (i-SEP, France) is an innovative filtration-based autotransfusion device able to salvage and wash both red blood cells and platelets. This study evaluated the device performances using human whole blood with the hypothesis that the device will be able to salvage platelets while achieving a erythrocyte yield of 80% and removal ratios of 90% for heparin and 80% for major plasma proteins without inducing signification activation of salvaged cells. Methods Thirty healthy human whole blood units (median volume, 478 ml) were diluted, heparinized, and processed by the device in two consecutive treatment cycles. Samples from the collection reservoir and the concentrated blood were analyzed. Complete blood count was performed to measure blood cell recovery rates. Flow cytometry evaluated the activation state and function of platelets and leukocytes. Heparin and plasma proteins were measured to assess washing performance. Results The global erythrocyte yield was 88.1% (84.1 to 91.1%; median [25th to 75th]) with posttreatment hematocrits of 48.9% (44.8 to 51.4%) and 51.4% (48.4 to 53.2%) for the first and second cycles, respectively. Ektacytometry did not show evidence of erythrocyte alteration. Platelet recovery was 36.8% (26.3 to 43.4%), with posttreatment counts of 88 × 109/l (73 to 101 × 109/l) and 115 × 109/l (95 to 135 × 109/l) for the first and second cycles, respectively. Recovered platelets showed a low basal P-selectin expression at 10.8% (8.1 to 15.2%) and a strong response to thrombin-activating peptide. Leukocyte yield was 93.0% (90.1 to 95.7%) with no activation or cell death. Global removal ratios were 98.3% (97.8 to 98.9%), 98.2% (96.9 to 98.8%), and 88.3% (86.6 to 90.7%) for heparin, albumin, and fibrinogen, respectively. The processing times were 4.4 min (4.2 to 4.6 min) and 4.4 min (4.2 to 4.7 min) for the first and second cycles, respectively. Conclusions This study demonstrated the performance of the SAME device. Platelets and red blood cells were salvaged without significant impact on cell integrity and function. In the meantime, leukocytes were not activated, and the washing quality of the device prevented reinfusion of high concentrations of heparin and plasma proteins. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New

Determination of irinotecan (CPT-11) and SN-38 in human whole blood and red blood cells by liquid chromatography with fluorescence detection

2003 ◽  
Vol 795 (2) ◽  
pp. 383-388 ◽  
Author(s):  
Floris A. de Jong ◽  
Ron H.J. Mathijssen ◽  
Peter de Bruijn ◽  
Walter J. Loos ◽  
Jaap Verweij ◽  
...  
Keyword(s):  
Liquid Chromatography ◽  
Red Blood Cells ◽  
Fluorescence Detection ◽  
Whole Blood ◽  
Blood Cells ◽  
Human Whole Blood

SCANNING ELECTRON MICROSCOPE: POSSIBILITIES FOR STUDYING KIDNEY DISEASES

2020 ◽  
pp. 176-182
Author(s):  
Mamaeva S.N. ◽  
Vinokurov R.R. ◽  
Munkhalova Ya.A. ◽  
Dyakonova D.P. ◽  
Platonova V.A. ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Electron Microscope ◽  
Scanning Electron Microscope ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Filtration Rate ◽  
Kidney Diseases ◽  
First Time ◽  
Scanning Electron

Currently, due to the intensive development of high-tech science-intensive medical and research devices, more and more attention is paid to the development of diagnostics of rare and difficult to diagnose diseases. It is known that among numerous nephropathies, hematuria may be the only symptom of kidney and urinary tract diseases, which complicates their diagnosis and treatment. In order to develop new approaches for the diagnosis of nephropathies, the authors have been studying the morphology of red blood cells in the blood and urine of children and adults using a scanning electron microscope for several years. The paper presents the results of studies of children with various kidney diseases, including IgA-nephropathy, and chronic glomerulonephritis. Scanning electron microscopy was used for the first time to detect nanoparticles on the surface of red blood cells, the size of which is comparable to the size of viruses, which became the basis for one of the authors ' assumptions, namely, the possible transport of certain types of viruses by red blood cells. Thus, some kidney diseases could be considered virus-associated. This paper presents for the first time the results of determining the glomerular filtration rate of both kidneys separately in the study of separate kidney function and of the study of urine smears obtained during catheterization of the ureters in patients with hydronephrosis of one of the kidneys by scanning electron microscopy. As in previous studies, nanoparticles were found on the surface of red blood cells, which leads to the conclusion about the possible viral nature of the disease of the considered patient. In addition, smear images obtained using a microscope showed a significant difference in the elements of the right and left kidneys urine, which did not contradict the data on the study of glomerular filtration rate. According to the authors, the capabilities of the scanning electron microscope can be applied in fundamental research of kidney diseases at the cellular and molecular levels, forming new ideas about their origin, as well as on the basis of which new methods of non-invasive diagnostics can be built.

scholarly journals An Evaluation of Morphological Changes and Deformability of Suspended Red Blood Cells Prepared Using Whole Blood with Different Hemoglobin Levels of Tibetans

2021 ◽  
pp. 1-10
Author(s):  
Rui Zhong ◽  
Dingding Han ◽  
Xiaodong Wu ◽  
Hong Wang ◽  
Wanjing Li ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Whole Blood ◽  
Blood Cells ◽  
Morphological Changes ◽  
Osmotic Fragility ◽  
Spherical Shape ◽  
Hypoxic Environment ◽  
Wide Range ◽  
Group A ◽  
Cell Membrane Protein

Background: The hypoxic environment stimulates the human body to increase the levels of hemoglobin (HGB) and hematocrit and the number of red blood cells. Such enhancements have individual differences, leading to a wide range of HGB in Tibetans’ whole blood (WB). Study Design: WB of male Tibetans was divided into 3 groups according to different HGB (i.e., A: >120 but ≤185 g/L, B: >185 but ≤210 g/L, and C: >210 g/L). Suspended red blood cells (SRBC) processed by collected WB and stored in standard conditions were examined aseptically on days 1, 14, 21, and 35 after storage. The routine biochemical indexes, deformability, cell morphology, and membrane proteins were tested. Results: Mean corpuscular volume, adenosine triphosphate, pH, and deformability were not different in group A vs. those in storage (p > 0.05). The increased rate of irreversible morphology of red blood cells was different among the 3 groups, but there was no difference in the percentage of red blood cells with an irreversible morphology after 35 days of storage. Group C performed better in terms of osmotic fragility and showed a lower rigid index than group A. Furthermore, SDS-PAGE revealed similar cross-linking degrees of cell membrane protein but the band 3 protein of group C seemed to experience weaker clustering than that of group A as detected by Western Blot analysis after 35 days of storage. Conclusions: There was no difference in deformability or morphological changes in the 3 groups over the 35 days of storage. High HGB levels of plateau SRBC did not accelerate the RBC change from a biconcave disc into a spherical shape and it did not cause a reduction in deformability during 35 days of preservation in bank conditions.

Glycophorin A-based exclusion of red blood cells for flow cytometric analysis of platelet glycoprotein expression in citrated whole blood

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Christina Berens ◽  
Johannes Oldenburg ◽  
Bernd Pötzsch ◽  
Jens Müller
Keyword(s):  
Red Blood Cells ◽  
Expression Analysis ◽  
Whole Blood ◽  
Blood Cells ◽  
Control Sample ◽  
Small Sample ◽  
Platelet Glycoprotein ◽  
Glycophorin A ◽  
Giant Platelets ◽  
Platelet Counts

AbstractObjectivesAnalysis of platelet glycoprotein (GP) expression by flow cytometry is applied for diagnostic confirmation of GP-associated thrombocytopathies. While platelet-rich plasma may be used for distinct identification of target events, this strategy is not feasible for small sample volumes or for patients showing low platelet counts and/or giant platelets. However, also the use of whole blood (WB) is hampered by the difficulty to discriminate platelets from red blood cells (RBC) in such patients. To circumvent these limitations, we evaluated the feasibility of a RBC gating-out strategy.MethodsIn addition to platelet GPIb, GPIIa/IIIa, as well as P-selectin (CD62P), citrated whole blood (CWB) samples were stained for RBC-specific glycophorin A (CD235a). CD235a-negative platelet events were further discriminated by forward-/side-scatter characteristics and platelet GP expressions analyzed relative to that of a healthy control sample processed in parallel.ResultsEstablished reference intervals allowed for clear identification of decreased GPIIb/IIIa- or GPIb expression pattern in samples of patients with confirmed Glanzmann thrombasthenia or Bernard–Soulier syndrome, respectively. It could be shown that the analysis of 2,500 platelet events is sufficient for reliable GP expression analysis, rendering the proposed method applicable to samples with low platelet counts.ConclusionsThis study demonstrates the feasibility of CD235a-based exclusion of RBC for platelet GP expression analysis in CWB. In contrast to direct staining of platelet-specific antigens for target identification, this indirect gating out approach is generally applicable independent of any underlying platelet GP expression deficiency.

Modified formulation of CPDA for storage of whole blood, and of SAGM for storage of red blood cells, to maintain the concentration of 2,3-diphosphoglycerate

Vox Sanguinis ◽  
2003 ◽  
Vol 85 (4) ◽  
pp. 253-261 ◽  
Author(s):  
P. A. Kurup ◽  
P. Arun ◽  
N. S. Gayathri ◽  
C. R. Dhanya ◽  
A. R. Indu
Keyword(s):  
Red Blood Cells ◽  
Whole Blood ◽  
Blood Cells
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