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White Blood Cells Function Pdf Download


The most important function of red blood cells (RBCs) is the carrying of oxygen, but they are also involved in inflammatory processes and during coagulation. RBCs are extremely deformable and elastic, as they are exposed to shear forces as they travel through the vascular system. In inflammatory conditions, and in the presence of hydroxyl radicals, RBCs loose their discoid shape. Here, ultrastructure of RBCs is studied using a scanning electron microscope, and we determine how fast changes in healthy individuals are noted after exposure to iron and glucose. We compare shape changes in these experiments to RBCs from diabetic and hemochromatosis patients (wild type, as well as hereditary hemochromatosis with mutations H63D/H63D, C282Y/C282Y, H63D/C282Y, C282Y/wild type and H63D/wild type). Thrombin is also added to whole blood exposed to iron, glucose and blood from diabetes and hemochromatosis patients. RBCs are easily deformed to a pointed shape in smears, and, with the addition of thrombin they are entrapped in the fibrin mesh of dense matted fibrin deposits. This entrapping causes severe shape changes due to the pressure of the fibrin onto the stressed cells. The most important observation of the current research is therefore how fast RBC can adapt in a changed environment and that the pressure of fibrin fibers may trap the RBC tightly in the resulting clot.




White Blood Cells Function Pdf Download



Red blood cells (RBCs) are discoid shape entities without a nucleus or mitochondria and their most important function is the carrying of oxygen to the cells of the body. They also play a fundamental role in the coagulation system and in inflammation [1], and has been used in biochemical studies to determine levels of antioxidants [2]. However, their role in inflammatory conditions is sometimes under-valued. Recently, Lopes del Almeida and co-workers pointed out that RBCs are not only a hemoglobin filled sacs, but are involved intimately in inflammation [1].


RBCs are extremely deformable and elastic, as they are exposed to shear forces as they travel through the vascular system [3]. These functions are highly dependent on membrane composition, and it is this composition that defines the properties of the RBC. Their membranes consist of 3 layers, the carbohydrate-rich glycocalyx on the exterior, the lipid bilayer that contains trans-membrane proteins and lastly, the membrane skeleton consisting of a structural network of proteins located on the inner surface of the lipid bilayer. In particular, the proteins of the membrane skeleton are responsible for the deformability, flexibility and durability and aids in recovering the discoid shape during rheology. The roughness of the cell membrane is also an indicator of cell's health state [4, 5] and the skeletal integrity of the membrane, measured as surface roughness, is well correlated to the functional status of the cell, with a decrease of the membrane roughness seen in cells from diseased individuals [3, 6]. Recently, researchers also reported that RBC distribution width (which is a measurement of the size variation as well as an index of the heterogeneity), is an easy, inexpensive, routinely reported test, and that may be used as significant diagnostic and prognostic information in patients with cardiovascular and thrombotic disorders [7].


Scanning electron microscopy (SEM) has also shown that in inflammatory diseases like thrombo-ischemic stroke and diabetes, RBCs have a changed ultrastructure [10, 17]. Previously, we have also shown that exposing RBCs of healthy individuals to iron, can also induce similar shape changes [10]. The changes were ascribed to the formation of hydroxyl radicals in experiments and the presence of these radicals in diseases conditions that ultimately lead to inflammation [18]. The question that now arises is whether ultrastructure can give us insights on how fast these changes may occur; and ultimately therefore, how adaptable these cells are. We know that RBC shape changes in diabetes, and when physiological levels of iron are added to healthy individuals. Here we determine if such changes are also seen in hereditary hemochromatosis. Also, we investigate if addition of glucose to healthy whole blood will induce changes to these cells, and compare results to when iron is added to healthy whole blood. The addition of iron and glucose was done to determine how fast RBCs can adapt in the presence of a changed environment. Additionally, as it is known that the coagulation system is changed in the presence of iron overload, due to the generation hydroxyl radicals in particular, we also compare RBC shape changes in the presence of thrombin, that creates fibrin networks and dense matted deposits in inflammation [19]. Therefore, RBC changes, in the presence of iron and glucose with the addition of thrombin, as well as in diabetes and hemochromatosis (also with and without the addition of thrombin) were studied and compared to that of RBCs from healthy individuals.


Same day processing of biospecimens such as blood is not always feasible, which presents a challenge for research programs seeking to study a broad population or to characterize patients with rare diseases. Recruiting sites may not be equipped to process blood samples and variability in timing and technique employed to isolate peripheral blood mononuclear cells (PBMCs) at local sites may compromise reproducibility across patients. One solution is to send whole blood collected by routine phlebotomy via overnight courier to the testing site under ambient conditions. Determining the impact of shipping on subsequent leukocyte responses is a necessary prerequisite to any experimental analysis derived from transported samples. To this end, whole blood was collected from healthy control subjects and processed fresh or at 6, 24 and 48 h after collection and handling under modeled shipping conditions. At endpoint, whole blood was assessed via a complete blood count with differential and immunophenotyped using a standardized panel of antibodies [HLADR, CD66b, CD3, CD14, CD16]. PBMCs and neutrophils were isolated from whole blood and subjected to ex vivo stimulation with lipopolysaccharide and heat-killed Staphylococcus aureus. Stimulated release of cytokines and chemokines was assessed by cytometric bead array. RNA was also isolated from PBMCs to analyze transcriptional changes induced by shipping. The complete blood count with differential revealed that most parameters were maintained in shipped blood held for 24 h at ambient temperature. Immunophenotyping indicated preservation of cellular profiles at 24 h, although with broadening of some populations and a decrease in CD16 intensity on classical monocytes. At the transcriptional level, RNAseq analysis identified upregulation of a transcription factor module associated with inflammation in unstimulated PBMCs derived from whole blood shipped overnight. However, these changes were limited in both scale and number of impacted genes. Ex vivo stimulation of PBMCs further revealed preservation of functional responses in cells isolated from shipped blood held for 24 h at ambient temperature. However, neutrophil responses were largely abrogated by this time. By 48 h neither cell population responded within normal parameters. These findings indicate that robust immunophenotyping and PBMC stimulated response profiles are maintained in whole blood shipped overnight and processed within 24 h of collection, yielding results that are representative of those obtained from the sample immediately following venipuncture. This methodology is feasible for many patient recruitment sites to implement and allows for sophisticated immunological analysis of patient populations derived from large geographic areas. With regard to rare disease research, this meets a universal need to enroll patients in sufficient numbers for immunoprofiling and discovery of underlying pathogenic mechanisms.


Cell-based functional immunological assays provide invaluable information both as diagnostic and prognostic biomarkers and as indicators of therapeutic efficacy in clinical trials. However, such assays require preparation and manipulation of viable leukocytes that are not activated or suppressed by the methods of collection, processing, or storage. Previous studies have suggested that a delay between time of collection and the preparation and cryopreservation of lymphocytes results in impaired function and decreased recovery1. Likewise, storing whole blood at 4 C has been shown to reduce leukocyte viability, function, and recovery following cryopreservation2,3,4,5,6, although other studies have observed relative preservation of phenotype7. On the other hand, storage at room temperature has been reported to have no effect on multiparametric immunophenotyping8 or viability9 but may alter subset representation2. Based on the need to perform ex vivo stimulation of isolated peripheral blood mononuclear cells (PBMCs) from patients at geographically disparate sites, we sought to clarify the effect of overnight shipping at ambient temperature on immunophenotype, transcriptional profile, and stimulated inflammatory cytokine and chemokine production, with an emphasis on monocytic responses.


PBMCs change scatter profile and become contaminated by low-density neutrophils over time. (A) PBMCs were isolated from whole blood using a density gradient (top panel). The forward and side scatter profile of the isolated PBMCs was altered as early as 6 h after collection; this effect was maintained through 24 h and largely reflected a change in size and granularity in lymphocytes (central population moved rightward). By 48 h the scatter profile of the PBMCs had collapsed leftward. In parallel, by 24 h the PBMC fraction showed a large increase in FSChiSSChi granulocytes, consistent with acquisition of a low density phenotype in neutrophils. (B) Neutrophils were enriched from whole blood using immunomagnetic negative selection (bottom panel). This population exhibited a marked dropout of FSChiSSChi cells as early as 6 h. Percents for relevant populations are shown.


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