Biomedical subjects
Publications and source records attributed to J Seghatchian.
During 2005 Norwegian Transfusion Medicine has faced some new problems, sought new solutions and gained strength. Interactions with the public and the health authorities have been important issues for consideration, which are discussed below.
Human plasma for therapeutic use, besides having optimal viral safety, must contain optimal levels of all coagulation factors and protease inhibitors to be clinically effective. Several new technologies for pathogen reduction of plasma (PRT) exist and are entering the stage of clinical testing. The main objective of this overview is to provide an update on the current states of three promising photoactive technologies that target pathogen nucleic acid for pathogen inactivation, applicable to single unit fresh-frozen plasma (FFP) and to highlight the experiences gained with classical pathogen reduction of pooled plasma using solvent-detergent (SD) treatment. It should be emphasized that none of the currently applied methods inactivate all types of pathogens and all have some effect on plasma quality when compared to fresh-frozen plasma. Pooled SD-plasma is the best documented clinical product, followed by methylene blue light treated (MBLT)-plasma. Recently, Psoralen light treated (PLT)-plasma has been introduced (CE-marked product in Europe) while Riboflavin light treated (RLT)-plasma is still under development. In principal, PRT for plasma not only differs in terms of the spectrum and log of pathogen reduction potential, but also in respect to the physicochemical/biological characteristics, and profiles of the adverse reactions, particularly in vulnerable patient groups. Therefore, an additional practical step such as oil extraction followed by chromatography to remove the solvent/detergent, and filtration or the use of some special absorbing matrix is required to reduce the residual photosensitive chemicals, their metabolites and photo adducts. This is required to improve the safety margin of the final product. Moreover, while it may be convenient to think that a combined pathogen reduction technology could improve the spectrum of known pathogens to be inactivated, one needs, in practice, to balance between the degree of pathogen reduction and the loss of some plasma protein activity. From the quality point of view, SD-plasma is a pooled standardized pharmaceutical product with extensive in-process control. However, both differences in production processes and the plasma source can influence final product quality. On the other hand, single unit plasma derived from nucleic acid PRT cannot be monitored by pharmaceutical process control and demonstrates the wide range of concentrations normally observed for plasma proteins. Pooling has the disadvantage that one single plasma unit can contaminate a whole pool, but this can be offset by several advantages that pooling and the SD process offer. Among these are reduction of a possible pathogen load by dilution and by neutralizing antibodies in the plasma pool, dilution and possible neutralization of antibodies and allergens which essentially eliminates transfusion-related acute lung injury (TRALI) and reduces allergic reactions significantly, removal of residual blood cells, cell fragments and bacteria, and removal of the largest von Willebrand-factor (vWF) molecules. On the other hand, some streamlining is required for technologies using single units of plasma, such as the use of plasma from male non-transfused donors to reduce TRALI and to avoid the O blood group in order to meet current specifications for FFP [Seghatchian J. What is happening? Are the current acceptance criteria for therapeutic plasma adequate? Transfus Apheresis Sci 2004; 31:67-79], and to exploit the potential benefit to inactivate residual lymphocytes and prevent transfusion-associated graft versus host disease. The cost effectiveness of pathogen inactivation is very low (> 2 million US dollar/life year saved), if however, non-infectious complications such as TRALI are taken into account; the cost for SDP is reduced to < 50,000 British pound/life year saved for those 48 years. Finally, from the therapeutic standpoint, two important questions still remain to be answered. First, whether the various pathogen reduced plasma products are clinically interchangeable and second, whether the conventional quality requirements of FFP are still adequate for the newer plasma products. These questions can only be answered by a head to head comparison, followed by large-scale clinical trials.
Important current issues in transfusion medicine in Norway are discussed. Current patient legislation specifically defines blood donors as patients, and blood and blood products are defined as drugs. Donor selection is controversial, especially deferral of all persons born in, or having lived for more than one year in areas with high prevalence of infections that are transmitted by blood. The threshold for becoming a blood donor is high, but registered donors donate frequently, e.g. 2,4 whole blood donations per year on average. Some blood banks have specialized in multicomponent aphereis technology, in particular collection of two units of red cells.
Cobe Trima version 4 is an apheresis system designed for the collection of combinations of Red Blood Cells (RBC), Platelets (PLT) and plasma components from a single donor. The validation of this apheresis system for PLT components in our institution evidenced the LRS efficacy, as leucoreduction was attained on a 100% basis. However, there were some unexpected occurrences regarding the PLT content. Certain donations showed large disagreements between programmed and obtained yields, not being found clear reasons for those outcomes. Furthermore, the mean platelet volume (MPV) was relatively low when compared with other platelets components produced by other methodologies. An investigation was initiated in order to know whether these shortcomings were donor or process/instrument related. A link was established between the raw material (donor), the process/instrument and the final product, and a new tool was introduced by the study of the LRS chamber. The LRS chamber content was assessed and the PLT cellular indices and PLT aggregation states compared with those obtained from the respective donor and the final product from the same origin. The storage stability of the final products was also analyzed based on these same tests to investigate if the initial low MPV had any deleterious effect during the shelf life of the components. Twenty five plateletpheresis donors, three of them new ones, were randomly selected for this study. For the first time, the content of the LRS chamber was used as a quality tool for identification of the cause of unexpected yields. Large aggregates remained in the LRS chamber in certain donors who were prone to undergo spontaneous aggregation, making the platelet yields low and platelet MPV very small. Based on repeated failures and on this new quality parameter showing LRS chamber abnormality, two donors were temporarily deferred from the panel. While it was not possible to identify the causes, it is appropriate to raise the question whether these profiles were Trima version 4 specific or not. Hence, further investigation is needed for a better clarification. In short, the simultaneous analysis of products and LRS content provided useful information not only for the characterization of donor-related phenomena but also helped in the identification of potential shortcomings in the machine performance allowing for remedial action to be taken on evidence based data.
BACKGROUND: Transferrin receptor mediates cellular uptake of iron, and the expression on cells reflects iron needs and erythropoietic activity. The results of measuring transferrin receptor in serum (sTfR) in blood donors are presented. STUDY DESIGN AND METHODS: Haemoglobin, serum-ferritin and sTfR were measured in 172 female and 174 male donors that had donated whole blood six or more times during the previous 3 years and in 96 female and 56 male new donors. RESULTS: Haemoglobin and sTfR were not significant different in new and repeat donors. New donors had significantly higher s-ferritin than repeat donors. Twenty donors had a Hb above the low limit for normal, but below the determined cut-off for donation. Only three of these had high sTfR and/or low serum-ferritin. Hence, of the total 492 donors 3.5% were below the Hb cut-off, but having Hb, s-ferritin and sTfR within normal ranges. 11.6% of new female donors belonged in this category. CONCLUSION: STfR is better than s-ferritin as a screening for iron deficiency. Most donors with low tissue iron neither have high sTfR, nor anaemia. There is probably no need to have a separate, higher than the lower normal range, requirement for Hb in donors. STfR measurements are probably most valuable in a setting where most donors are repeat donors.
Both red blood cells and platelets undergo lesions upon storage which affect their function and possibly their clinical outcome. Some of these lesions are reversible, others not. Improved additive solutions and leukocyte depletion can delay the appearance of storage lesions. In addition, cellular apoptosis leads to numerous mitochondrial and surface changes during storage which have the potential to induce immune suppression by tuning down the innate immune system. This overview highlights some laboratory and clinical aspects of red cell and platelet storage lesions.
The establishment of the Norwegian Fractionation Project (Project) was of major importance in preserving national self-sufficiency when plasma, cryoprecipitate and small batch factor IX-concentrates were replaced by virus inactivated products in the last part of the 1980s. Fractionation was performed abroad by contract with Octapharma after tenders on the European market. All Norwegian blood banks (>50) participated in the Project. Total yearly production was 50-60 tons of mainly recovered plasma. From 1993 solvent detergent (SD) treated plasma has replaced other plasma for transfusion. The blood banks paid for the fractionation and/or viral inactivation process, while the plasma remained the property of the blood banks and the final products were returned to the blood banks. The Project sold surplus products to other Norwegian blood banks and the majority of the coagulation factor concentrates to The Institute of Haemophilia and Rikshospitalet University Hospital. Both plasma and blood bank quality was improved by the Project. Clinical experience with the products has been satisfactory and self-sufficiency has been achieved for all major plasma proteins and SD plasma, but a surplus exceeding 3 years consumption of albumin has accumulated due to decreasing clinical use.The Project has secured high yields of the fractionated products and the net income from the produced products is NOK 1115 (140 Euros or US dollars) per litre plasma. An increasing surplus of albumin and the possibility of significant sales abroad of currently not fractionated IVIgG, could lead to a reorganisation of the Project from that of a co-ordinator to a national plasma handling unit. This unit could buy the plasma from the blood banks and have the plasma fractionated by contract after tender, before selling the products back for cost recovery. The small blood banks could produce plasma for products for the Norwegian market, while surplus products from the larger blood banks which are certified for delivery of plasma for fractionation of products to be consumed in the European Community, could be sold on the international market.
The distribution of leucocyte subpopulations in platelet concentrates (PC) derived from pre-storage filtered platelet-rich plasma (PRP), the cell suspension obtained by reverse filter washing and the post-filtered PC, were monitored by immunophenotyping analysis using CD3, CD20 and CD33. Leucocyte activation analysis with the CD11b marker revealed that this molecule is up regulated in neutrophils taken from the filter. This, together with the loss of cell viability during the enrichment process, suggests that contact with the filter matrix and processing and storage of samples containing leucocytes may lead to activation and loss of leucocyte viability. These changes were found to be more pronounced in less stable myeloid cells and account for the differences reported among various authors which in some cases related to operational conditions such as the enrichment process used and the length of time between filtration and analysis of samples. Finally, statistical analysis of the results obtained by immunophenotypic studies indicate that post-filter samples (S) contain significantly higher numbers of CD33+ myeloid cells when compared to (PF) the pre-filter samples (65.03%+/-12.6 and 24.56%+/-14.73, p<0.0000), with a decrease in T cells (50.72%+/-14.80 in PF and 24.05+/-9.48 in the cell suspension (S), p<0.0007) and B cells (14.96+/-9.31 in PF and 9.9+/-5.22 in S, p<0.201). A new strategy for assessing the influence of the filtration process on residual leucocyte activation and viability is described. This has direct relevance to collection, processing, storage and quality monitoring of PC.
To investigate the pro- and anti-coagulant properties of platelet-derived microvesicle (MV), a novel procedure for the removal of plasma proteins was designed, which allows determination of these activities in a purified system in the same reaction vessel. While the pro-coagulant activity of the isolated MV increased on day 5 as compared to day 1, the anti-coagulant activity was significantly reduced.
Plasma and red blood cell quality are affected both by citrate concentration and the levels of extracellular leukocyte and platelet derived substances, accumulated during storage of blood. The effect of leukocyte filtration on the storage stability of whole blood was therefore studied in blood collected in standard CPD and 0.5CPD (CPD with half strength citrate concentration). A total of 52 units, 12 of them with reduced citrate concentration, were leukocyte-filtered with Pall( whole blood filter (WBF1 or 3). No differences in leukocyte or platelet reduction were observed with the two citrate concentrations. However, with 0.5CPD a significantly longer filtration time and increased complement activation was observed. The effect of pre-storage leukocyte filtration on the plasma quality of whole blood was therefore only studied with standard CPDA1 anticoagulant solution (normal strength citrate concentration). Leukocyte filtration did not affect the von Willebrand factor concentration, while a small reduction (7%, p=0.04) in factor VIII (FVIII) concentration was observed. During storage, however, FVIII decreased more slowly in the filtered than in the unfiltered product, and, from day two, the FVIII content was significantly higher in the filtered product (46% versus 30% at 28 days, p<0.001). Factor V (FV) demonstrated a 16% reduction (p<0.001) upon filtration, followed by an additional 8% in the next 24 h and only a 4% reduction the next 27 days, while unfiltered products demonstrated a continuous reduction to 26% at 28 days. While the beta-thromboglobulin (beta-TG) concentration significantly increased (from 836 to 2483 IU/ml, p<0.001) during leukocyte filtration, no further increase was observed during storage. In contrast, unfiltered products demonstrated an increase to 5762 IU/ml (p<0.001) at 14 days, followed by a slight, not significant, reduction. This indicates platelet activation during filtration and explains a parallel reduction in FV. Filtration induced no increase in prothrombin fragment 1+2, while a slight increase was observed in some unfiltered products after 28 days of storage.Pre-storage leukocyte depletion thus improves the coagulation factor content of plasma in stored whole blood.
182 full double donations were collected, using 7-9 cycles with an in-house guideline based on donor HCT, and platelet count. 95.6% of donations were within specifications for platelet yield and 100% were leucodepleted. To overcome the potential for red cell spill-over when the platelet peak is too small, automated continuous monitoring of machine performance has been introduced reducing the potential for spill-over. To understand the impact of the continuous exposure of platelets to the filter, the quality of the first and second part of the collection was monitored using a new set of markers for the platelet storage lesion (activation, microvesiculation, cellular injury, complement and response to ADP). The results were equivalent and compared well with routine practice. Our new protocol allows a wide range of donors to be selected and the highest percentage of products to meet the UK specification with consistency. It is recommended that it can be used for double dose platelets with routine SPM.
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.
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.
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. MATERIALS AND METHODS: Initially in the National Blood Service (NBS) England, protocols were standardized on the use of LeucoCount reagents with either Becton-Dickinson or Beckman Coulter flow cytometers. RESULTS: Standardization of protocols resulted in a decreased intersite variability of red cell samples (from 36% to 9% at a level of 11 and 10 cells/ micro l, respectively), and 100% of sites (n = 11) fulfilled the validation criteria. However, we also evaluated the use of alternative reagents with the result that reagents from either Becton-Dickinson or Beckman Coulter, used on either a Becton-Dickinson or Beckman Coulter flow cytometer, passed our validation criteria. CONCLUSIONS: It is critical to include samples from filtered products containing white blood cells in validations of leucocyte enumeration methodology, as results may differ between methods using these samples but not using spiked or fixed material. Standardized gating strategies and optimization methods for flow cytometers are critical for obtaining equivalent results with different reagents and instruments.
The 21 presentations have been arranged into two broad inter-related groups: (i) Quality of therapeutic preparations and studies on viral/bacterial inactivation procedures. (ii) Diagnostic assays for viral safety and quality control of blood components. Furthermore, in line with the main theme of this symposium, I have attempted to provide an overview of pertinent continuing investigation of blood component safety and supply, the "twin pillars" of transfusion therapy, focusing on the three pointers (3Ps) of the quality system-patient, process and product.
The relationship between the platelet storage lesion (PSL) and programmed cell death (apoptosis) is poorly understood. Nevertheless, there is some experimental evidence that platelets contain most of the components of the apoptosis machinery and both the apoptotic process and the PSL lead to platelet activation and microvesiculation with expression of phosphatidyl serine (PS) on the outer layer of cell membrane, a hallmark of all nucleated cells. The PS exposure is believed to contribute to the development of inflammatory or immunomodulation process, to the regulation of haemostatic balance and the ultimate clearance of dead or fragmented cells from the circulation. While there is no doubt that apoptosis, as a form of genetically encoded programmed cell death in nucleated cells, is triggered by several signalling stimuli at the nuclear level, there is some doubt as to whether platelets, as enucleated cells have retained the memory of the "parental" megakaryocytes for apoptosis or whether platelet mitochondrial DNA has a major role in both the apoptotic process and the PSL. The storage lesion occurs during processing and storage subsequent to mechanical trauma, hypoxic conditions or exposure to cold. In this brief report some observational evidence is provided in support of the notion that the PSL and apoptosis may be related to each other, despite the fact that, in contrast to the 'parental' megakarocyte, the platelets appear to survive upon stimulation with a high concentration of protein kinase inhibitors such as staurosporine (STS), in the presence of cycloheximide (CHX) which inhibit protein synthesis. This is a model which is often used to regulate the level of survival signals. The possible relevance of platelet microvesiculation to transfusion practice is briefly discussed.
With the increasing demand for leukoreduction of blood components and the implementation of universal leukoreduction in several countries, the problems associated with leukocyte filtration of sickle cell trait blood have been revisited. Currently, there is no unified standard practice for sickle cell trait donors. Different blood centers adopt different policies. While some defer these donors from red cell component donation, some do not. Some screen all ethnic African donors for hemoglobin S (Hb S), others do not. Furthermore, there are differences in views of whether sickle cell trait red cells should be considered as equivalent to non-sickle cell trait red cells. Some blood centers do not give red cells from a sickle cell trait donor to the newborn or patients undergoing general anesthesia as a preventative measure. In this presentation, we discuss the epidemiology of the sickle gene, the sickling process, problems associated with leukoreduction of sickle cell trait whole blood and red cells, and some unresolved issues concerning donor referral and the usage of sickle cell trait blood.
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