Red cell storage lesion assessed by the levels of potassium, haemoglobin and Annexin V in supernatants.

The conventional and a new marker of global cellular lesion (Annexin V) are used to assess the processing/storage-induced changes in four types of RBC and filtered blood at 4 degrees and 22 degrees C, stored for a period of 35 days, in multi-satellite packs. It appears that mechanical trauma and presence of leucocytes and residual platelets have potential to increase levels of all markers of storage lesion, but to a variable extend. We have also provided new evidence that multi-satellite packs can be safely used for up to 35 days for small volume transfusion to sick premature infants, in a well-managed system by administering several transfusions from the same donation, hence reducing donor exposure.

Validation of different enrichment strategies for analysis of leucocyte sub-populations: development and application of a new approach, based on leucofiltration.

Characterisation of the nature of leucodepletion failure, based on the analysis of leucocyte sub-populations, is an essential task for continual improvement in the clinical safety of blood components and for the selection of a safe and appropriate leucodepletion process. Such information is also critical for improving the quality of filters through an understanding of the mechanism of leucocyte removal filters. Unfortunately, the residual leucocytes of filtered blood components are around the sensitivity of current leucocyte-counting technologies, hence a need for an enrichment process. This preliminary report deals with the merits of various WBC enrichment strategies for the analysis of WBC sub-population subsets. A new procedure, based on refiltering and backflushing the content, with up to 30-fold enrichment, is described. This one-step procedure has the advantage of concentrating both native and atypical leucocytes seen in blood components. The latter may account for lack of interchangeability of results obtained by various counting methods.

A national quality assessment scheme for counting residual leucocytes in unfixed leucodepleted products: the effect of standardisation and 48 hour storage.

BACKGROUND: WBC counting, an essential part of quality monitoring of WBC-reduced blood components, is carried out logistically within 48-72 h of collection. The between-laboratory variability and effects of 24-48 h storage were investigated using three major counting technologies. STUDY DESIGN AND METHODS: Samples of RBC and platelets with WBC in the range 0-50/microl were transported by courier. WBC counting was performed on days 1 and 2, by IMAGN 2000, flow cytometry and Nageotte, initially using local protocols and then using a national flow protocol. Up to 15 laboratories participated in each exercise. RESULTS: For "real failed leucodepleted" red cell products, higher levels of variability were observed for flow and Nageotte, as compared to IMAGN. For spiked RBC samples at critical decision making point (3-20 WBC/microl), between-laboratory the coefficients of variation (CVs) were low for IMAGN and were the highest for Nageotte. Flow cytometry CVs were generally high but improved subsequent to standardisation of sampling and the gating strategy. A similar pattern in the variability of results was observed for platelet concentrates. Sign tests using all samples (carried out for each method in each exercise; 25 in total) demonstrated no overall tendency for larger WBC counts to be recorded on day 1 when compared to day 2, although this difference was significant (p < 0.001) in certain cases depending on the nature of the spiked product. CONCLUSIONS: We conclude that while a good performance is achieved using validated automated technologies for low residual leucocyte counting, the unification of reagents and standardisation of sampling and gating strategies are essential in obtaining interchangeable results. Unfixed RBC and platelet samples can generally be stored for 48 h before WBC counting.

A platelet quality assessment scheme for comparing the performance of quality monitoring laboratories in the UK National Blood Service.

This exercise focused on performance of NBS quality monitoring establishments with respect to enumeration of low leucocyte and other quality indexes of platelet concentration. Paired identical leucodepleted platelet samples, spiked with WBC (20 cells/microl) in 'vacuette' or 'pouch' were assessed by participants (n = 20) on days 1, 2 and 5. For low WBC counting, all laboratories gave estimates within acceptable range (+/-25%) and good agreement between storage and assay methods was observed on days 1 and 2. Day 5 results showed greater variability. Under improved performance criteria (+/-15%), only one laboratory under-estimated at days 1 and 2. Similarly, other parameters demonstrated good agreement between storage methods on days 1 and 2. At day 5, mean results were often significantly different to previous days. Improved performance target (+/-15%) will allow identification of non-conformers.

The effects of leukodepletion on the generation and removal of microvesicles and prion protein in blood components.

BACKGROUND: Universal leukodepletion (LD) has been implemented in the United Kingdom to reduce the risk of transfusion-transmitted variant Creutzfeldt-Jakob disease. If LD causes microvesiculation of blood cells, however, potentially infectious membrane-associated prion could reach the final products. STUDY DESIGN AND METHODS: We have measured microvesicles (MV) derived from red cells (RBC-MV), platelets (PLT-MV), and white blood cells (WBC-MV) and cellular prion protein (PrP(c)) in blood components produced by four whole-blood, five RBC, three PLT, and two plasma LD filters and three plateletpheresis techniques. RESULTS: RBC-MV and PLT-MV were either unaltered or reduced by all processes, with PLT-MV reduced 10-fold by RBC LD and greater than 300-fold by plasma LD. WBC-MV were reduced or unchanged by RBC and PLT LD and reduced by plasma LD. Whole-blood filtration appeared to increase MVs derived from granulocytes, but the load in the final components was comparable to that in processed RBCs in additive solution. PrP(c) was reduced by whole-blood, RBC, and plasma LD and unchanged by PLT techniques. There were differences between various filters and techniques, which were generally minor compared to the overall effects. CONCLUSION: These findings suggest no detrimental effects of LD processes in terms of generation of MVs or PrP(c) release.

Variables determining blockage of WBC-depleting filters by Hb sickle cell trait donations.

BACKGROUND: Sickle cell trait donations can block leukodepletion (LD) filters or fail to LD, but the variables affecting blockage are unclear. STUDY DESIGN AND METHODS: To identify critical variables for further study, the relationship was investigated between filter blockage and donor characteristics, processing conditions, PLT and coagulation system activation, and microvesicle formation in donations with (n = 63) and without (n = 40) sickle trait. With eight filter types whole blood was LD either at ambient temperature on Day 0 or after overnight 4 degrees C hold. Markers of PLT activation (CD62P and CD63 expression and soluble CD62P) and coagulation activation (activated FXII and prothrombin fragment 1 + 2 [F1 + 2]), RBC microvesicles, blood gases, and residual WBCs were measured. RESULTS: All Day 0 filtrations blocked (n = 7). On Day 1, no filter tested was 100 percent successful, with most achieving an approximate 50 percent success rate. Two filters blocked consistently and an additional filter did not block, but resulted in 50 percent of units with high residual WBC counts (30 x 10(6)-394 x 10(6)/unit). Day 1 filtration was not improved if performed at 4 degrees C. Donor RBC variables and prefiltration measures varied little between blocked and successful filtrations except pO2, where 9 of 17 blockages had a pO2 of less than 5.0 kPa, compared with 0 of 13 completed filtrations. F1 + 2 levels increased after filtration in sickle trait units, a consequence of slow flow rate. CONCLUSION: Filter blockage in sickle trait donors cannot be predicted by donor characteristics or filter type and is not related to PLT or coagulation activation, but can be reduced by storing units at 4 degrees C before filtration.