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Characterization of Buffy Coat-Derived Granulocytes for Clinical UvA-DARE (Digital Academic Repository) Neutrophil defects and deficiencies van de Geer, A. Publication date 2020 Document Version Other version License Other Link to publication Citation for published version (APA): van de Geer, A. (2020). Neutrophil defects and deficiencies. General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) Download date:01 Oct 2021 Chapter 6 1 Characterization of buffy-coat-derived granulocytes for clinical use: a 2 comparison with granulocyte-colony stimulating factor/dexamethasone- pretreated donor-derived products. 3 Annemarie van de Geer1, Roel P. Gazendam1, Anton T.J. Tool1, John L. van Hamme1, Dirk de Korte1,2, Timo K. van den Berg1, Sacha S. Zeerleder3,4 and Taco W. Kuijpers1,5. 4 1. Dept. of Blood Cell Research, Sanquin Research, Amsterdam, The Netherlands 2. Dept. of Product and Process Development, Sanquin Blood Bank, Amsterdam, The Netherlands 3. Dept. of Immunopathology, Sanquin Research, Amsterdam, The Netherlands 4. Dept. of Hematology, Academic Medical Center, University of Amsterdam, The Netherlands 5. Dept. of Pediatric Hematology, Immunology & Infectious disease, Emma Children’s Hospital, Academic Medical Center, University of 5 Amsterdam, The Netherlands Vox Sang. 2017 Feb;112(2):173-182 6 7 8 9 10 A Chapter 6 AbstracT background and objectives Buffy coat-derived granulocytes have been described as an alternative to the apheresis product from donors pretreated with dexamethasone and granulocyte-colony-stimulating factor (G-CSF). The latter is – dependent on the local and national settings - obtained follow- ing a demanding and time-consuming procedure, which is undesirable in critically ill septic patients. In contrast, buffy coat-derived products have a large volume and are often heavily contaminated with red cells and platelets. We developed a new pooled buffy coat-derived product with high purity and small volume, and performed a comprehensive functional characterization of these granulocytes. Materials and methods We pooled ten buffy coats following the production of platelet concentrates. Saline 0.9% was added to decrease the viscosity and the product was split into plasma, red cells and a “super” buffy coat. Functional data of the granulocytes were compared to those obtained with granulocytes from healthy controls and G-CSF/dexamethasone-pretreated donors. Results Buffy coat-derived granulocytes showed adhesion, chemotaxis, reactive oxygen species production, degranulation, NETosis and in-vitro killing ofStaphylococcus aureus, Escherichia coli, and Aspergillus species comparable to control and G-CSF/dexamethasone-derived granulocytes. Candida killing was superior compared to G-CSF/dexamethasone-derived granulocytes. Immunophenotyping was normal, especially no signs of activation in the buffy coat-derived granulocytes were seen. Viability was reduced. Buffy coats are readily avail- able in the regular blood production process and would take away the concerns around the apheresis product. Conclusion The product described appears a promising alternative for transfusion purposes. Key words: neutrophil, granulocyte, transfusion, buffy coat, G-CSF, dexamethasone 142 Buffy coat – derived granulocytes for clinical use IntrODUctiON Life-threatening bacterial and fungal infections are a major problem in patients using medi- cations with immunosuppressive effects, like chemotherapy. Reasons are intensified che- motherapy regimens leading to prolonged severe neutropenia and rising resistance against antimicrobial agents. This results in mortality rates up to 90% in patients with invasive fungal infections.1-3 Administration of donor granulocytes in addition to antimicrobial drugs to patients with refractory infections may be a last resort, but data from large randomized controlled trials demonstrating its benefits are lacking. Nevertheless, there are data sug- gesting less infection-related mortality after granulocyte transfusions4-8. To obtain enough granulocytes for donation, donors are pretreated with granulocyte-colony-stimulating fac- tor (G-CSF) and dexamethasone, or with steroids alone9 for granulocyte mobilization from the bone marrow prior to leukapheresis (from the G-CSF ánd dexamethasone pretreated donors: “GTX”). We have previously shown that these granulocytes function well10, although there is a significant partial in-vitro Candida killing defect11. Also, donor recruitment may be demanding, entails the risk of too few available donors and can be considered unethical in countries where patients are responsible for their own donor recruitment. 6 Dependent on the local and national settings, donors undergo extensive medical investiga- tion before pretreatment and are allowed to donate up to 4-5 times, whereas the recipients usually need more transfusions. To improve separation of blood components during leuka- pheresis low amounts of Hydroxyethyl Starch (HES) are used in many countries entailing the risk of fluid retention, coagulopathy and allergic reactions12,13. Much higher volumes of HES have been proven to cause higher morbidity and mortality rates in critically ill patients and are therefore not used in a clinical setting anymore14,15. To bypass any of these concerns, granulocytes from buffy coats (BCN) might be an alternative source for transfusion. BCN are readily available in the blood production process, and there is already some evidence that BCN are an appropriate alternative to GTX16. This study provides a new BCN product, with high purity and small volume. It builds upon work of Bashir et al16 to use granulocytes from pooled buffy coats for transfusion and by our knowledge, it is the first to offer an extensive comparison between the functionality of BCN, GTX and control granulocytes. We found that BCN functions are equivalent to those of GTX and controls. Materials and MethoDs Pooled buffy coat preparation, GTX donor pretreatment Thrombocyte concentrates were produced from overnight stored whole blood (room tem- perature) derived from healthy voluntary donors at the Dutch national blood bank, Sanquin. 143 Chapter 6 After this, buffy coat pools from 5 different donors were left. Two of these pools were merged (according to the methods as described by others17) and 250 ml Saline 0.9% was added to decrease the viscosity and improve blood component separation. The suspension was centrifuged (700g, room temperature, 4 minutes and 15 seconds) and separated in red cells, a ‘super’ buffy coat (our transfusion product) and a supernatant, with a Compomat G5 system (Fresenius Kabi, Bad Homburg, Germany). Granulocytes isolated from the ‘super’ buffy concentrates were used for the experiments. Heparinized blood samples from GTX donors were obtained from different donors by venipuncture at the moment of donation. These donors had received 5 µg/kg G-CSF sub- cutaneously and 8 mg dexamethasone orally 12-16 hours prior to donation. Heparinized control blood samples were collected by venipuncture from healthy volunteers at Sanquin. Buffy coats and samples from GTX donors were obtained from volunteer donors at Sanquin after written informed consent was obtained. Control samples were obtained via an internal system at Sanquin after informed consent was given and after consulting the Medical Ethical Committee from the Academical Medical Center Amsterdam. All procedures are conducted in accordance with the 1975 declaration of Helsinki as revised in 2013. Granulocyte isolation and cell counts We have isolated granulocytes from the ‘super’ buffy coat concentrates and from the heparinized blood samples from GTX donors at time of donation and controls. These cells were used for all experiments and will be called BCN-derived, GTX-derived, and control granulocytes. Our previous studies have shown that there is no difference in results as to whether we isolate granulocytes from the GTX product bag or after a separate venipuncture (data not shown). Granulocyte isolation was performed with a Percoll gradient of 1.076 g/ml for the GTX and controls and 1.074 g/ml for the BCN concentrates, as this last cell suspension is 18 hours old. Cells were centrifuged (800g, 21°C, 20 minutes) and erythrocytes in the pellet were lysed twice with isotonic NH4Cl-KHCO3-EDTA solution at 4°C. Thereafter, the solution was centri- fuged (500g, 4°C, 5 minutes) and cells were resuspended in incubation medium (132 mM NaCl, 6 mM KCl, 1 mM CaCl2, 1 mM MgSO4, 1.2 mM potassium phosphate, 20 mM HEPES, 5.5 mM glucose and 0.5% (w/v) human serum albumin, pH 7.4) on room temperature. Purity of the collected granulocytes was >95%, as judged by cytospin. Granulocyte counts were determined with an automated cell counter (Sysmex XT-2000iV, Kobe, Japan). Samples were taken before and
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