J Seghatchian

The effect of leucocyte depletion on the quality of fresh-frozen plasma

The aim of this study was to evaluate the quality of leucodepleted (LD) fresh‐frozen plasma (FFP) produced using one of five whole blood filters (Baxter RS2000 & RZ2000, NPBI T2926, Macopharma LST1 and Terumo WBSP) or two plasma filters (Pall LPS1 and Baxter FGR7014). Whole blood or plasma was filtered within 8 h of collection at an ambient temperature. Samples were taken pre‐ and post filtration for analysis of coagulation factors and complement activation (n = 7–12 for each type of filter). All filtered units (209–286 ml) contained < 5 × 106 residual leucocytes and < 30 × 109/l platelets. Statistically significant losses of factors V, VIII, IX, XI and XII and increases in markers of coagulation activation were observed (0–21%), which were dependent on filter type. None of the filters had a significant effect on von Willebrand factor (VWF) multimeric distribution or the activity of VWF and factors II, VII or X. The effect on levels of C3a appeared to be related to the filter surface charge: positively charged filters resulted in C3a generation, whereas negatively charged resulted in C3a removal. None of the observed changes are likely to be clinically significant unless subsequent processing of plasma (such as pathogen inactivation) results in further losses of coagulation factors.

Evaluation of Cobe Trima for the collection of blood components with particular reference to the in vitro characteristics of the red cell and platelet concentrates and the clinical responses to transfusion

This study evaluated Cobe Trima for donor and operational acceptability, the quality and storage stability of the blood components collected, and the clinical responses to transfusion. The study was carried out in 2 phases; phase 1 assessed the efficiency of red cells and platelet collection, and the characteristics of the components collected before and after storage. Phase 2 was an evaluation of operational issues and the in vitro characteristics of the red cells and platelet concentrates at the time of transfusion in respect to their cellular content, and leucocyte (interleukin IL-6 and IL-8) and platelet-derived (Rantes) cytokine levels. Cytokine levels were also measured in the donors before and after the collection procedure and in patients both before and after transfusion. The clinical responses to a small number of transfusions were assessed. The Cobe Trima was found to be straightforward to use by the operators, although additional operator training was required to manage occasional uncertainty with alarm messages. It was acceptable to the donors except for the occurrence of citrate reactions in 3/6 donors in phase 1; this problem persisted in phase 2 (6/15 donors), and needs to be addressed in the future. All blood components met UK product specifications apart from 2 platelet concentrates, 2 red cell concentrates, and one unit of FFP; the red cell and platelet concentrates had good storage characteristics. The 2 procedures, which resulted in low platelet yields, were due to occlusion of the plasma line; the method for installation of the harness has been subsequently modified to prevent this. 2 red cell concentrates showed haemolysis; the reason for this was not established. The Factor VIII level was satisfactory in plasma and the cellular content was low. The responses to 12 platelet transfusions were expected as in a group of haematology patients, and no immediate adverse effects were reported with any of the transfusions. Leucocyte-associated (IL-8 and IL-6) and platelet-associated (Rantes) cytokine levels were not elevated in donor samples taken before or after the collection procedure, or in the red cell and platelet concentrates at the time of issue. Pre- and post-transfusion IL-8 levels were raised in one patient with non-immune platelet refractoriness, and normal in 2 patients with excellent or almost satisfactory responses to platelet transfusions raising the question as whether IL-8 could be used as a laboratory marker for non-immune platelet refractoriness due to infection.

Effect of filtration, storage and platelet suspension media on platelet indices.

We describe a new approach for assessing filtration-induced changes in cellular indices of platelet concentrates at the beginning and the end of storage, using pairs of identical packs. The results revealed that post-filtered products did not store as well as their counterparts. Filtration did not induce any significant changes on aggregation as determined by spontaneous aggregation nor was there a disparity between leucocyte peroxidase/basophil count. We recommend filtration on day 2 which causes minimal loss of platelets and less change in the mean platelet volume.

Studies on the improvement of leucodepletion performance of the Haemonetics MCS+ for production of leucodepleted platelet concentrate

With the implementation of universal leucodepletion, an in-line, negatively charged LRF6H leucodepleting filter became an essential part of the Haemonetics MCS+ plateletpheresis system. A larger-scale (968) study using the standard protocol revealed a 2.79% leucodepletion failure rate (standard < 5 2 10 6 leucocytes per adult therapeutic dose). Factors influencing the efficacy of the filter were investigated. The pH of the filtrate was 7.0, the temperature 28°C and filtration rate 80 ml/min. Reduction of the filtration rate to 30 ml/min (784 doses) reduced leucodepletion failure to 0.38%. Measurement of the leucocyte count, pre- and post-filtration of the platelet products, revealed that donations from 1% of donors contained substantially larger numbers of leucocytes in pre-filter samples (300-1500/ w l) than in control samples (35-70/ w l). This number tends to increase progressively with subsequent donations in these individuals, leading to leucodepletion failure, whilst peripheral leucocyte counts remain normal. The new continuous filtration protocol (version C) using a less impact filter LRF-XL and a lower (7 ml/min) head pressure was also effective but failure still occurred twice on one of the donors who persistently showed high pre-filter count. We conclude that leucodepletion failures in the Haemonetics system are related to both donor leucocyte (i.e., being light and non-adherent) and operational/filter performance.

The development of a national standardized approach for the enumeration of residual leucocytes in blood components.

Background and Objectives The UK Blood Transfusion Services implemented universal leucocyte depletion of the blood supply in November 1999. To provide statistical process monitoring of these processes, automated methods were introduced to count residual leucocytes (white blood cells) in blood components.

Current position on preparation and quality of leucodepleted platelet concentrates for clinical use

Double dose leucodepleted PC without filtration is considered to be the most cost-effective way of preparing leucodepleted PC in a reasonable time. The procedure lends itself to a multicomponent system and production of hyperconcentrate and dry platelets, with < 10-15 ml plasma in final product and viral inactivation without considerable loss of in vitro platelet functions. Platelet concentrates obtained by various procedures are highly heterogeneous, even if a standard protocol is used for the preparation. Therefore, standard/standardisation in both production and testing procedures remain a challenging area in order to obtain comparative results. Attention needs to be focused on growing and complex technical features of preparation and on the use of new filter material in terms of biocompatibility and the related effects of activated factors on function of platelets and leucocytes. Both the production process and storage containers appear to contribute to various cellular lesion and generation of some biological response modifiers such as complements, cytokines and microparticles. In this respect it is relevant to adopt a multiparameter analysis for the validation of platelet quality as some markers of platelet storage lesion have different affinity to various surfaces, leading to false under estimation. Further development work is still needed in preparation and usage of dry platelet, platelet alternative and bacterially safe products. The underlying conditions of the transfused patients is also an important issue in this respect, it is interesting to note that patients with high IL8 levels have a substantially lower platelet recovery.

Studies on the characterisation of the cause of leucoreduction failures, with particular reference to extra gatal events

The causes of leucodepletion failure are multifactoral and can be related to haematological variability in blood donors or donation, defective filters, poor specimen handling or ageing, and/or the presence of non-adhering leucocyte/platelets. Since refiltering removes all types of leucocytes, including the populations appearing as extra gated events, we have developed a practical method for refiltering the failed leucodepleted components on standard filters and back-flushing the second filter to assess the nature of the WBC sub-population. In practice, recovered leucocytes from red cell filters and whole blood mainly consist of neutrophils. Those from platelet and plasma filters were mainly lymphocyte with considerable differences depending on the type of leucodepletion process. Atypical leucocytes are often seen in some pre-/post-cellular leucofiltered components. These appear characteristically as small WBC with a lower affinity for filter matrix, or as cell fragment, pinched leucocyte or apoptotic cells. Different reagents in use show variable sensitivity in identifying these extra gatal events. Storage of leucodepleted samples also induces different types of abnormality in leucocyte dot plot. A useful practical approach for characterisation of the nature of leucocyte sub-populations causing failure in leucodepleted components is provided.

THE UK STRATEGY FOR MONITORING UNIVERSAL LEUCODEPLETION

This summary manuscript deals with the NBS approach to quality monitoring of universal leucodepletion based on representative sampling. There is also a brief discussion on the failure rate and potential causes for the inter site variations in white cell enumeration by current methods.