Novel Automated Preparation and Cold Storage of Buffy-coat- Derived Platelets in Additive Solutions: in Vitro Studies

This thesis focuses on (i) the in vitro quality of platelets (PLTs) prepared by novel automated techniques using the OrbiSac and the Atreus 2C+ systems; and (ii) evaluation of the ability to store PLTs for a prolonged time at 4oC in an attempt to optimize the conditions under which PLTs are prepared and stored. There has been an increase in the demand for supportive PLT transfusion therapy and in the search for a procedure for preparation of PLTs from pooled whole blood (WB)derived buffy coats (BCs) for transfusion. Until now, this technique, which includes a sequence of manual steps, has been laborious and non-standardized, and has resulted in significant variance in PLT yield. This has led us to evaluate a novel automated system (OrbiSac) for the preparation of PLTs from BCs derived from WB and a novel automated system (Atreus 2C+) for preparation of BCs from WB, by studying PLT counts, and recovery and storage effects. The results of our in vitro studies suggest (i) that the OrbiSac technique is equivalent to the standard manual method regarding in vitro PLT characteristics during storage for 7 days, with uniform recovery of PLTs; and (ii) that PLTs derived from BCs produced using the novel automated Atreus 2C+ system to separate either fresh WB or WB stored overnight are equivalent to PLTs prepared using a semi-automated fresh WB separation process. Work detailed in this thesis (Papers I and IV) demonstrates that the Atreus 2C+ and the OrbiSac systems used in combination allow for automated production of PLTs with maintenance of in vitro PLT quality during 7 days of storage. The development of the processing systems (Papers I and IV) was followed by an attempt to tackle the problem of how to refrigerate PLTs for transfusion (Papers II and III). Platelets are traditionally stored at 22oC, which facilitates bacterial growth, and bacterial sepsis is regarded as the main risk of transfusion-transmitted diseases. For this reason, PLT storage is limited to 5 days. Storage at 4oC would reduce not only the risk of bacterial growth, but may also delay the impairment of PLTs. Since the 1970s, considerable improvements have been made concerning processing and storage of PLTs, resulting in longer survival and better function, and providing an option for prolonged storage at 4oC. Work detailed in this thesis demonstrates (i) that PLTs stored without agitation at 4oC largely maintain their metabolic and cellular characteristics for 21 days of storage. We confirm that they lose their discoid shape and show that this loss of discoid shape during storage at 4oC is associated with reductions in metabolic rate, and a decreased release of α-granule content. Furthermore (ii), we demonstrate that cold-induced activation, occurring at 4oC, is not associated with increased expression of PLT membrane proteins and activation markers during long-term storage. Our findings concerning the ability of cold temperatures to preserve PLT quality and prolong the storage period under modern blood bank conditions suggest that cold storage of PLTs may be possible in the future. However, this ability of PLTs to circulate and function in vivo remains to be demonstrated. LIST OF PUBLICATIONS This thesis is based on the following publications, which are referred to in the text by their Roman numerals I-IV. The publications are reproduced in this thesis with kind permission of the publishers. I. Larsson S, Sandgren P, Sjödin A, Vesterinen M, Gulliksson H. Automated preparation of platelet concentrates from pooled buffy coats: In vitro studies and experiences using the OrbiSac system. Transfusion 2005 May;45(5):743-