Elsevier ; : p. Medical Physiology for Undergraduate Students. Updated: May 30, Accessed: June 3, Johnston A, Uetrecht J. Current understanding of the mechanisms of idiosyncratic drug-induced agranulocytosis.. Expert Opin Drug Metab Toxicol. Drugs in the aetiology of agranulocytosis and aplastic anaemia.. Eur J Haematol Suppl. Filgrastim treatment of three patients with clozapine-induced agranulocytosis.. J Clin Psychiatry. Use of granulocyte colony-stimulating factor in the treatment of methimazole-induced agranulocytosis: a case report.
Clinical Case Reports. Textbook of Human Physiology for Dental Students. Textbook Of Practical Physiology - 2nd Edn. Mast cell: A multi-functional master cell. Front Immunol. Textbook Of Medical Physiology. Elsevier India ; Travlos GS. Toxicol Pathol. The impact of chronic intermittent hypoxia on hematopoiesis and the bone marrow microenvironment. All you need to know about bone marrow.
Updated: December 15, Accessed: June 6, Stem Cells. Frontiers in Endocrinology. Major medications with a definite association with agranulocytosis. Last updated: January 1, Accessed: May 7, Quinto et al. Relationship between haemoglobin and haematocrit in the definition of anaemia. Tropical Medicine and International Health. McClain, Michael L. A Practice of Anesthesia for Infants and Children. Having established the enrichment of leukocytes by LSA-3, we explored the utility of adapting this approach to clinical practice.
The results of 51 experiments with clinical blood samples consisting of 26 cases of cell enrichment using the pushing style method, as well as 25 cases using the pulling style method, were found to be consistent with the results of this study. The serial improvements in the design of the LSAs models result in an approximately fold gain in the leukocyte yield of LSA-3, where the leukocyte-to-erythrocyte ratio increases from to S1 and S2 Tables. Specific conditions used to produce the enrichment data are described in S1 and S2 Tables in the Supporting information section.
The novel biomedical microdevice, utilizing centrifugation without introducing any chemical contamination media density gradient or antibodies , significantly improves analysis of blood [ 23 ]. However, the cytoplasm of leukocytes has become blurred, and cell morphology has already changed by cell lysis. This novel and facile approach to blood sampling required optimization of several methodological parameters that were shown to affect cell capture efficiency.
These include the LSA geometry design, the position at which the buffy coat localizes in the upper funnel, and the optimal duration of the second centrifugation. All these factors facilitate enrichment of leukocytes from whole blood. This work documents and validates the present device design and identifies the parameters that optimize separation and yield of homeostatic cells.
This technology provides new ideas and methods which was shown a label-free, size and density-based separation of blood cells with high throughput based on the localization of the buffy coat cells within a narrowly defined band occupying the upper funnel of LSA-3 designs.
Based on these validation studies, the advantage of the present modifications to earlier LSA designs can be explained because of conserving extremely high leukocyte populations that were lost in earlier versions of the design.
In LSA-1, Due to the outlet graphic structure of the main body taking the shape of a trumpet, leukocytes were found to adhere to the inner wall of narrow end the trumpet under the pressure applied during anticlockwise rotation the threaded-booster, resulting in failure to reach the enrichment target.
Whole blood was naturally layered in LSA-2 by application of centrifugal force, and large numbers of leukocyte cells could pass through the central channel and to form the buffy coat within the outlet upper funnel. While this design showed improvement in the leukocytes recovery rate of LSA-2 over LSA-1, bottleneck jams of cellular traffic occurred because the diameter of the central channel of LSA The central channel diameter of LSA-3 was larger than LSA-2 so that the resistance of the cell exchange was reduced, where an increased diameter of the central channel of the LSA-3 produced a significantly higher mean cell recovery rate and greater efficiency of cell separation.
Localization of the buffy coat layer within the outlet upper funnel after the first centrifugation step is important for achieving a maximal cell enrichment outcome. With this criterion, high throughput and capture yield can be achieved by both the pushing and pulling methods. We speculate that in the earlier devices, a small number of leukocytes attached to the walls of the upper funnel, which caused cell loss.
In these studies, both the pushing and pulling methods of cell displacement in LSA-3 can be used to enrich leukocytes, with no significant difference in cell yields between the two options. The original intention of employing both methods was to let the researcher or clinician choose the method that was more workable and user-friendly.
Based on the present studies, our results show that while a second centrifugal step is required to complete leukocyte isolation and transfer, its duration length does not have much effect on capture yields. Accordingly, we recommend that 2 min be allocated as the centrifugation time to accomplish optimal enrichment in the shortest time. In summary, we report 3D printed several biomedical microdevices that perform label-free, high-throughput enrichment leukocytes from non-diluted whole blood by centrifugation.
The novel LSA-3 microdevice utilizes 3D printing designs that improve enrichment efficiency by displacing the buffy coat layer farther away from the band of erythrocytes. The novel microdevice design requires no ancillary pumping mechanism nor expensive disposables to operate and may become a viable candidate for a standardized and streamlined initial isolation protocols in clinical laboratories. In recent years, the development of microfluidic technology is more and more rapid, but there are still many challenges, such as the interference of a large number of erythrocyte cells in the process of blood sample analysis [ 25 ].
Therefore, numerous assays require removal of erythrocytes from whole blood as an essential step to analysis of clinically-relevant cells. The novel LSA-3 biomedical microdevice in this study accomplishes depletion of most erythrocytes from whole blood with good cell morphology and activity, which has the huge potential to be used as a valuable sample preparation tool in both research and clinical settings.
The novel LSA-3 biomedical microdevice also have some potential limitations. Future work will focus on improving the leukocyte-to-erythrocyte ratio and providing better standardize procedures for laboratory use. For example, further modifications of the diameter of the central channel may eliminate the initial dynamic reduction of leukocyte concentration caused by the impeded traffic of leukocytes during their entry into the upper funnel.
In addition, height of the funnel could be altered to affect better separation efficiency of leukocytes. Furthermore, a higher height aspect of the funnel could result in a smaller residual volume of erythrocyte, thereby improving separation efficiency. We appreciate the assistance of Ming-jian Wei-ye Technology Co.
Chongqing, China during the experiments. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract Leukocytes have an essential role in patient clinical trajectories and progression.
Introduction Enumeration of leukocyte cells within peripheral blood provides valuable clinical information to physicians about the status of a patient. Design of the leukocyte sorting accumulators The structure of leukocyte sorting accumulator LSA have an overall uniform length of 70 mm and a diameter of 12 mm, including the main body, the threaded-booster, and the threaded-cap Fig 1.
Download: PPT. Fig 1. Schematic diagram of the leukocyte sorting accumulators. Enrichment of leukocytes by the leukocyte sorting accumulator We tested the three types of LSA design with samples consisting of 2—3 ml of the undiluted whole blood that were introduced into the main chamber to enrich leukocyte.
The buffy coat formed when the sample was processed in the first centrifugation step S1C Fig. A syringe conical is next connected to the outlet of the LSA Plus, with a anticlockwise rotation to push the threaded-booster of the main body, you can see clearly that most of the plasma and buffy coat is transferred to the syringe under positive pressure S1D Fig.
Finally, it is collected and cells were counted on automatic hemocytometer, including erythrocyte cell and leukocyte cell count. A significant difference exist among individuals with normal-range of hematocrit, so that the buffy coat was formed at an uncertain position of the lower funnel after the first centrifugation step S2C Fig.
The buffy coat is going to move relatively from the lower to the upper funnel of the LSA-2 under positive pressure by anticlockwise rotating the threaded-booster of the main body S2D Fig. It is collected cells from the upper funnel for complete blood cell count.
It is clearly that the buffy coat was formed at an uncertain position of the lower funnel for the first centrifugation step Fig 2D. If the observed shape of the buffy coat layer is intact on the upper funnel, it will be collected into an empty tube for complete blood cell count.
Otherwise, it is necessary to ensure integrity of the buffy coat by the second centrifugation condition. Fig 2. Processing of leukocyte extraction by LSA-3 the pushing style method.
The buffy coat is formed at an uncertain position of upper funnel at the first centrifugation conditions Fig 3D. It is very ingenious to clockwise rotate the threaded-booster of the main body to cause the buffy coat moving down to a specific location within the upper funnel under negative pressure Fig 3E. If the buffy coat layer has been broken up in the process, the second centrifugations will be required to coalesce the layer.
Finally, it is collected from the upper funnel that cells were counted on automatic hemocytometer, including erythrocyte cell and leukocyte cell count. Fig 3. Processing of leukocytes by LSA-3 designs pulling style method. Optimization of pushing and pulling protocols by the LSA-3 device for enrichment of leukocytes The study further considered the effect of position of the buffy coat layer in the upper funnel when the LSA-3 device is used and a second centrifugation step is required, followed by either a pushing style or pulling style of translocation.
Fig 4. Schematic illustration of the working principles of the LSAs. Traditional centrifugal blood cell As a control experiment, the blood cells were centrifuged at g for 10min. Cell lysis Isolation of total leukocytes using erythrocyte lysis buffer was performed by mixing 1 ml of whole blood with 14 ml of lysis buffer NH 4 Cl buffer for 5 min. This correction calculation had a more substantial effect on other cytokines. This study has also revealed other classes of cytokines that are associated with RBCs as outlined in Table 4.
In a recent study, RBCs were identified as a major source of the nuclear protein IL, which is part of the IL-1 cytokine superfamily They found that levels of IL increased in the plasma of sickle cell patients who had experienced varying levels of haemolysis. In addition to those already mentioned, a range of growth factors, chemokines, and pro- and anti-inflammatory cytokines were also identified Tables 1 — 3.
A small number of chemokines are known to bind to and localise on the RBC membrane through ligation of DARC 9 , 15 , however a detailed investigation in the literature of how the remaining cytokines are partitioned in RBCs is yet to be conducted.
We sought to identify which, if any, of the cytokines were associated with the membrane fraction of RBCs. To achieve this, the cytokine profile of the cell lysates containing the cell membranes was compared to the cytokine profile of the cytosolic fraction lacking the cell membranes.
There was a significant lower amount of four cytokines in the cytosolic fraction Fig. Cytokines in RBC lysate and cytosol. Level of cytokines in the total lysate or cytosolic fraction of RBCs as measured by Bio-Plex and reported as fluorescence.
It was of interest in this study to investigate if RBCs were able to bind these cytokines. As indicated in Fig. RBCs have been observed to sequester IL-8 out of whole blood, and subsequently inactivate the chemokine, until cellular saturation through ligation of DARC Our data support these findings and suggest that the RBCs sequestered at least a portion of the recombinant cytokines out of solution. Incubation of recombinant cytokines with RBCs. To determine if assaying the RBC lysates could have been confounded by sample interference, the recovery efficiency of RBC lysates was determined by spiking in known concentrations of recombinant cytokines and measuring the final yield.
Quantitative data collected for these two cytokines should be used with caution. The recovery efficiencies reported here are similar to reported values for plasma assayed on the Luminex platform 19 , Belabani et al.
Using that criteria, 38 of the 48 cytokines analysed in this study fell within the acceptable range for RBC lysates. Even so, detectable levels of 46 of the 48 cytokines were present in the conditioned media for one of more biological replicates Fig. The aim of this experiment was to identify and quantify which cytokines would be released from RBCs in the absence of anything that could stimulate a negative feedback loop, such as the presence of plasma.
Plasma contains a variety of cytokines, and differing levels of these are likely to impede the release of cytokines. In addition, to ensure the RBCs were not lysing during this time, haemoglobin levels were monitored and determined to be minimal as outlined below. The detectable concentration of IL-8 varied substantially between biological replicates; the range of IL-8 in the conditioned media was 2.
The limitations of immunoassays in detecting complex bound cytokines have been previously described Furthermore, there is evidence that some molecules are contained within RBCs in an inactive form so that they are ready for rapid release Oonishi et al. They theorised that the molecules were not being manufactured, but instead that enzymes were converting the molecules into a form that then rendered them active and detectable. The concentrations of released cytokines are summarised in the Supplementary results Supplementary Table S3 — S4.
Analysis of RBC conditioned media has not been widely reported in the scientific literature, but its effect on T cells and fibroblasts has been briefly reported 8 , However, the cytokine profile of the conditioned media in these studies was not investigated.
We hypothesised that the cytokine profile of the RBC conditioned media could be manipulated by excessive sample processing, we thus tested the effect of multiple wash cycles on the release of cytokines from intact RBCs. Cytokines in RBC conditioned media. Amongst the proteomic analyses of RBCs, no studies have yet identified the presence of cytokines as reported in this study. There are likely to be a number of reasons for this such as issues with the dynamic range of the proteins; the abundance of these cytokines is approximately million-fold lower than the abundance of haemoglobin in RBCs.
In addition, the detection of low abundance proteins will be hindered by the vigorous washing procedures typically done during proteomic sample preparation. As an example, in a report on the analysis of the proteome of RBC membranes, the isolated cells were centrifuged, filtered, washed four times in an isotonic buffer, and finally the cells were lysed and the resulting membranes were washed another four or five times This processing method may be optimal for the identification of transmembrane or structural proteins, but in the identification of cytokines bound to the surface of the cell membrane this is unlikely to be effective.
To study the effect of aggressive sample handling, we washed intact RBCs ten times in an isotonic buffer, which significantly attenuated the release of six cytokines into the RBC conditioned media Fig. Notably, these results were observed without a significant change in RBC purity Changes in the cytokine profile following extensive processing has similarly been observed for platelets wherein, excessive washing or vigorous processing techniques have been shown to promote their degranulation and subsequently an increase in the secreted detectable cytokine levels Although the platelet literature is clear on the effects of processing, the only other enucleate cells in humans, RBCs, are regarded as transcriptionally and translationally inert and incapable of secretory activity.
Cytokines in cell lysates and conditioned media following cell washing. The mean concentration of haemoglobin in the conditioned media samples corresponded to approximately 1. Unlike any other study to date, the concentrations of cytokines in RBC lysates have been quantified and reported according to their relative abundance in whole blood Tables 1 — 3.
The results of this study revealed that 46 of the 48 cytokines assayed were detected in RBC lysates of healthy volunteers Tables 1 — 3 and for 42 of these cytokines, they were more than 4-fold higher in the RBC lysates than in the plasma with a median fold increase of This panel of cytokines identified in RBCs cover a range of pro- and anti-inflammatory factors, chemokines, and growth factors.
A number of these cytokines are regularly monitored in plasma or serum for biomarker studies 1 and the expression and secretion of these cytokines are thought to be limited to specific subsets of white blood cells The identification of these cytokines associated with RBCs indicates that an additional, previously unknown, level of cytokine interactions may exist in blood.
In clinical studies, optimal sample preparation is crucial for reproducible results To achieve this, techniques generally focus on minimising white blood cell and platelet activation during plasma or serum isolation.
Haemolysis of 0. Thus, an undetectable amount of haemolysis would quickly confound results of a single assay and introduce inter-sample variability. After incubation, a wide range of cytokines were detected in the conditioned media of RBCs Fig.
The concentration of haemoglobin in the RBC conditioned media indicated lysis of 1. For 43 cytokines, the concentration in the conditioned media was higher than what could be attributed to the RBC lysis. This suggests that the proteins were being released or shed in a mechanism that is independent of haemolysis. Very few studies have determined the content or activity of RBC conditioned media.
One study reported that T cell proliferation was stimulated in the presence of RBC conditioned media and that this stimulation was comparable with intact RBC-mediated proliferation 23 and another observed that culturing fibroblasts in the presence of intact RBCs or RBC conditioned media stimulated the secretion of IL-8 and the expression of the corresponding mRNA 8.
As is typical of the literature on RBCs, the cytokine profile of the conditioned media was not investigated, let alone quantified, in any of these studies. When normalised for cell number, the concentration of 28 of the cytokines in the conditioned media were higher than that detected in the RBC lysates prior to incubation.
The cytokine with the largest difference between pre-culture lysate concentrations and the concentration in the conditioned media was IL-8 Tables 1 — 3 and Fig. With white blood cell correction, IL-8 was almost undetectable in the RBCs lysates prior to incubation 6.
The lack of detectable IL-8 in the RBC lysates was unexpected as it is one of the few cytokines that has been identified by multiple reports as being present in and released from RBCs 15 , 23 , An increase in secreted cytokines from stored RBCs is generally attributed to white blood cell contamination, but a study suggested otherwise That study compared the cytokine release after storing whole blood for 42 days to RBCs and leukodepleted RBCs 29 and they reported that the storage of whole blood resulted in the lowest concentration of secreted IL-8 1.
These results suggest that perturbation of these cells stimulated the release of IL In light of this study, it is likely that the reason for the increased cytokine concentration in the stored RBC conditioned media is controlled release by intact RBCs. Free IL-8 has a serum half-life of minutes, however, studies have shown that there is a dynamic equilibrium between monomers, dimers, and cell-surface bound forms of IL-8 Although, in principle, RBC lysis releases the cytosolic contents for analysis, proteins such as IL-8 may be released from lysed RBCs in a bound form that renders them inaccessible to immunoassay.
Using PBS for the production of the conditioned media is a limitation of this study, as it is not directly relevant to what will be occurring in vivo. Future studies in understanding the dynamic processing occurring between plasma and RBCs would be of interest and could be achieved using labelled cytokines or cells.
This study revealed that washed RBCs have significantly attenuated release of a variety of cytokines Fig. Notably, washing RBCs appears to have some therapeutic benefit in transfusion medicine. In fact, washing RBCs following storage appears to be correlated with reduced incidence of adverse events and in some cases, improved outcomes such as in mice with haemorrhagic shock One of the reported benefits of this procedure is the depletion of inflammatory proteins from the blood pack This depletion is likely achieved by both removal of the storage media which contains inflammatory proteins 31 , and also by the removal of loosely bound cytokines from the surface of RBCs, as observed in this study Fig.
Cholette et al. This effect was represented by resulting lower serum levels of inflammatory cytokines and a change in trend towards reduced mortality However, there appears to be a delicate balance required in the amount of washing needed to achieve this therapeutic benefit.
Excessive washing increases RBC osmotic fragility and can lead to increased haemolysis following transfusion which can cause downstream adverse effects 33 , 34 , The results of this study may have implications on understanding the pathology of haemolytic conditions such as sickle-cell disease.
In these disorders, the increase in free haemoglobin in the plasma has been attributed to causing complications such as platelet activation and inflammation 36 , This causes the particles within the blood to separate, making them easier to collect.
Before the centrifugation process begins, the proper precautions must be taken. First, the correct type of tube for the blood will need to be determined. If an EDTA tube is used, the blood will need to be processed within two hours of being collected from the donor. Additionally, the sample could contain diseases, contaminants, or infections.
For this reason, it is imperative that proper safety precautions be taken when preparing for buffy coat extraction. Once all proper procedures have been followed, the sample is placed in the centrifuge machine, ensuring that the sample is balanced and properly secured. The temperature of the unit, as well as how fast it will need to be spun, will depend on the type of tube used and the volume of the sample. Typically, the spinning process lasts approximately 10 minutes.
Using a pipette, the now concentrated leukocyte layer aka the buffy coat is extracted from the top of the centrifuged sample. The amount of buffy coat collected will be approximately 20 percent of the original whole blood sample.
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