Pranee Krailadsiri

Annexin V, a new marker of platelet storage lesion: correlation with dMPV.

Released annexin V, an intracellular platelets glycoprotein, was used to determine the cellular injury which occurred during storage of platelet concentrates. Twenty-eight units of leuco-reduced apheresis platelet concentrates, obtained without leucocyte filtration, were analysed. Released annexin V showed a significant correlation with EDTA-induced shape changes of platelet (r = 0.62, P < 0.01) while poor correlation was found between released annexin V and pH or MPV. The combination of released annexin V with dMPV provides excellent markers of the platelet storage lesion for quality monitoring, based on morphological/functional integrities and cellular injury, which are of direct relevance to clinical efficacy of platelet concentrates.

The quality of methylene blue treated FFP and cryo

It is currently unclear to what degree methylene blue in combination with removal, of cells from plasma, by filter, can directly influence the loss of active components of plasma and whether the co-precipitation of FVIII/vWf with fibrinogen/fibronectin is affected by combined methylene blue and light treatment (MBLT). These questions are investigated using the Fenwal system. Our results indicate that up to 15% of the FVIII and IX are lost due to exposure of plasma to filters and methylene blue (MB). The illumination leads to a further 10-15% loss of all other major clotting factors. Factor XI appears to be highly sensitive to the MBLT-process, while inhibitors of the coagulation system are less affected. MBLT did not grossly influence the distribution of fVIII/vWf:Ag between cryoprecipitate and cryosupernatant using a paired control/test protocol, although the fVIII/vWf recovery is reduced in MBLT samples. The three commercially available MBLT processes differ in terms of operational aspects. These may have some impact on overall quality/safety and bioequivalency.

Preparation and storage characteristics of white cell‐reducedhigh‐concentration platelet concentrates collected by anapheresis system for transfusions in utero

BACKGROUND: Important concerns with regard to in utero platelet transfusions are avoidance of volume overload and the immunomodulatory effects of residual white cells (WBCs). This study evaluated a modification of a leukocyte‐reduction system (LRS, Spectra, COBE BCT) for apheresis, which collects high‐concentration WBC‐reduced platelets (HCPs) for in utero transfusion.

State-of-the-Art-Review: Microvesicles in Blood Components: Laboratory and Clinical Aspects

There is ample evidence for the presence of microvesicles (MV) of different sizes and functions in various blood components. A variety of mechanisms have been proposed for the formation of MV. These include mechanical injury, shear stress, cell activation, activation of complements, hypoxia, and the cell aging process. While MV share many biological properties and surface receptors of their parental cells, they demonstrate significant differences in membrane asymmetry of the inner membrane phospholipid, in particular phosphatidylserine (PS). This provides high-affinity binding sites for the components of the prothrombinase complex. To what extent these MV contribute to hemostatic effectiveness, immudomodulation, and some untoward effects of the transfused blood components remains to be fully elucidated. Several methods for qualitative and semiquantitative characterization of MV are now available. Although in most cases it is necessary to separate MV from the intact cells for improved characterization, recent advances in flow cytometry make it possible to accurately differentiate MV in the presence of their parental cells on the basis of light scattering and fluorescent intensity. This review focuses on four main areas of MV in blood components: (1) the proposed mechanisms of platelet vesiculation, (2) factors influencing the formation of MV, (3) laboratory analysis of MV, and (4) the clinical impact of the presence of MV in blood components. Key Words: Microvesicte—Vesicutation—Biood component—Ptatelets—Transfusion.

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.

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.

Cytokines as quality indicators of leucoreduced red cell concentrates

Different types of filters are currently used for leucodepletion of red cell concentrates. These filters meet the specification for leucoreduction (<5 x 10(6) leucocytes/ATD) but the quality of the final product may differ depending on the performance of the filters for effective removal of both leucocytes, platelets and possibly cytokines which are associated with transfusion reactions. We measured the levels of three representative cytokines: IL-8, RANTES and TGF-beta1 in red cell concentrates prior to and subsequent to the filtration procedure on day 1 and after a storage period of 35 days. Low levels of IL-8 (10-24 pg/ml) in the control unfiltered concentrates on day 1 which increased by approximately twofold on storage. Filtration reduced the levels of IL-8 on day 1 and day 35, in filtered concentrates in comparison with their control unfiltered counterparts. Leucoreduced concentrates produced by three different filters showed similar IL-8 levels on day 1 and day 35. However, concentrates prepared using another type of process showed a twofold increase in IL-8 levels on storage in comparison with day 1. None of the concentrates tested contained any detectable RANTES and TGF-beta1 suggesting a minimal platelet content. These results indicate that a combination of IL-8, RANTES and TGF-beta1 are useful quality indicators for validation of leucoreduced red cell preparations.

Residual red cell and platelet content in WBC-reduced plasma measured by a novel flow cytometric method.

BACKGROUND The levels of residual red blood cells (RBC) and platelets (PLT) in WBC-reduced plasma are often below the lower detection limit of automated blood cell counters. This study established a novel flow cytometric method for the enumeration of residual RBC and PLT in plasma. Furthermore, their levels in WBC-reduced plasma prepared by using various filters were investigated. MATERIALS AND METHODS WBC-reduced plasma was prepared from two sources: (i) filtration of buffy-coat reduced plasma using dock-on Baxter, Pall and Maco Pharma plasma filters; (ii) filtered whole blood using integral Asahi RZ2000, Maco Pharma LST1, NPBI, and Pall WBF2 whole blood filters. Residual RBC and PLT counts were assessed by using a TruCount tube (Becton Dickinson) containing a known number of lyophilized fluorescent beads. RBC and PLT were labelled with dual monoclonal antibodies, anti-CD41-R-phycoerythrin and anti-glycophorin A-fluorescein isothiocyanide, and analyzed by flow cytometer. RESULTS The flow cytometric method used in this method can detect residual RBC, PLT as well as RBC-MV simultaneously. The sensitivity of the assay was 50 x 10(6) cells/l with the coefficient of variations < or = 10%. Baxter and Maco Pharma plasma filters consistently reduced both RBC, RBC-MV and PLT to below 50 x 10(6/)l. Plasma derived from day 1 RZ2000 filtered whole blood contained PLT below 50 x 10(6) cells/l, whereas day 0 NPBI filtered whole blood showed the highest level of residual PLT. CONCLUSION A sensitive and accurate method for the detection of low levels RBC, RBC-MV, and PLT was established to measure their levels in WBC-reduced plasma. The procedure is simple and practical for routine quality monitoring of plasma, as well as for setting a new specification for WBC-reduced plasma.

Counting of residual WBCs in WBC-reduced blood components: a multicenter evaluation of a microvolume fluorimeter by the United Kingdom National Blood Service

BACKGROUND: Implementation of universal WBC reduction of blood components means that automated analytical methods may be the only satisfactory way for production laboratories to meet increased testing requirements.

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.