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Abstract

  1. Top of page
  2. Abstract
  3. Critical parameters during the donation process and in the period before separation
  4. Leucodepletion
  5. Whole blood separation and component preparation
  6. Pathogen inactivation
  7. Production of cellular blood components from undifferentiated cells in vitro
  8. Disclosures
  9. References

In the past three decades, the production of standard blood components from whole blood donations and from apheresis collections has reached a widely accepted high technical standard, paralleled by semi-automated methodology to safeguard the stability of the production process and an internationally harmonized pharmaceutical quality. More recent methodological advances include pathogen inactivation as well as novel separation methods, which are challenging the previous achievements. The current review aims (1) to summarize the current status of implementation of novel techniques into the routine preparation process of blood components, (2) to discuss upcoming approaches in the area of component preparation, (3) to identify clinical needs that justify novel investigations into blood component quality and (4) to point out the status on the production of standard blood components by in vitro differentiation from stem and progenitor cells in vitro.


Critical parameters during the donation process and in the period before separation

  1. Top of page
  2. Abstract
  3. Critical parameters during the donation process and in the period before separation
  4. Leucodepletion
  5. Whole blood separation and component preparation
  6. Pathogen inactivation
  7. Production of cellular blood components from undifferentiated cells in vitro
  8. Disclosures
  9. References

The production process begins with the selection of suitable donors. For standard blood components, minimum haemoglobin concentrations have been recommended in most guidelines worldwide (e.g. > 12·5 g/dl in women and > 13·5 g/dl in men) to assure adequate haemoglobin contents in the final red cell concentrate; a literature search however failed to reveal significant published data. Other physical properties of donors’ red cells have so far not been considered. Further parameters that likely affect the quality of blood components, including the geometry and flow dynamics of needles and blood bag tubing systems, the function of valves and their location at critical points in the multiple-bag systems, as well as the anticoagulant and stabilizer solution composition, may all influence product quality; however, this is an area of little publication activity, and clear statements cannot be made from the literature.

Hygiene regimens have to be in place to adequately counteract contaminations such as from skin-resident bacteria in the blood. In addition to secure that the disinfection of the donor arm is performed adequately, many services and a number of countries have introduced 100% inlet-line diversion of blood flow (i.e. the first 20–40 ml of a donation are diverted into a separate pouch, later used to filling the test-tubes) as a measure to reduce accidental contaminations of blood products. Enforcement of side deviation has lead to reductions in bacterial contamination in both whole blood donations and apheresis in the order of 40–88% [1,2]. Diversion together with improved donor arm disinfection has improved the percentage of reduction in contamination from 47% to 77% [2]. In addition, systematic measures have to be in place to safeguard optimal hygiene also in the donation areas; again, published evidence is lacking. We know that many donor services have introduced release procedures for any mobile donation site before starting a donation session and have validated the use of appropriate disinfectants and surveillance methods. Table 1 lists some main current questions in the area of component production.

Table 1.   Current issues in the production of standard blood components
ParameterEnd-point(s)References (examples)
All components
Storage time and temperature of whole blood before separationQuality and function of blood components pharmaceutical effectors; side-effectsWilsher et al., 2009; [4,6,15,50]
Red cell concentrates
Storage timeClinical outcome – older vs. younger RCs[51]
Proinflammatory changes (cytokines, microvesicles)[21,22]
Platelet concentrates
Additive solutionsImpact on in vivo PLT function[25–28]; for review: [23]
Storage timeImpact on platelet function and accidental bacterial contamination[34–37]
Concentrated (‘dry’) plateletsFunctionality?[24]
Platelet countsEndoreplication in platelets during storage?[52]
Therapeutic plasma
Time-point after donationQuality, coagulation factor activities[5]
HLA/HNA antibodiesPrevention of TRALIEder et al., 2007

Temperature control

Manufacturing blood components along a defined GMP process includes the control of the product and ambient temperature. Blood is naturally cooling upon entering blood bag systems, when mixing with the anticoagulant that is usually kept at room temperature. Although specific limits for this have not been systematically analysed, two main procedures have been followed for whole blood: (1) hold of the blood on cooling plates and (2) maintenance in special temperature-controlled transport boxes or containments installed in the vans that shuttle to mobile donation sessions.

Storage time and temperature preseparation

Some groups have begun to analyse effect of overnight storage of whole blood on the quality of the ensuing red cells, platelets and therapeutic plasma components. Gulliksson and van der Meer [3,4] reported that using a range of blood bag systems from different manufacturers, the red cell concentrates from overnight-stored whole blood, in comparison to 8-h storage preseparation, contain significantly lower levels of extracellular potassium, 2,3-diphosphoglycerate (2,3-DPG) and pH, and higher ATP. Cold storage (4°C), in comparison to storage at room temperature, resulted in higher haemolysis rates in the red cell concentrates (analysed at the 8-h preseparation whole blood storage time-point). Wilsher et al. have before this study reported similar differences, concluding that both methods yielded acceptable quality red cell concentrates and plasma quality parameters [5]. Serrano et al. [6] analysed effects of overnight vs. 8-h storage on plasma quality and found that in all parameters their pharmaceutical quality fulfilled the necessary release criteria, although significantly lower in FII, FVII, FVIII (72%), F IX, FX, FXI, and von Willebrand factor were found, as well as higher levels of fibrinogen and antithrombin III. Heiden et al. [7] reported analogous developments. Effects on platelet quality were observed and included the generation of white-blood-cell fragments in leucoreduced platelet concentrates (PCs), which increase up to levels that are equivalent to the amounts of intact WBCs that induce HLA immunization (i.e. > 5 × 106/unit) when products are stored prior to filtration [8].

Phagocytosis and self-sterilization

Another reason to pursue the maintenance of freshly drawn blood at ambient temperature is the ability of phagocytes in fresh blood to eliminate accidentally contaminating bacteria by phagocytosis. This is an accepted and widely pursued practice specifically enforced after introduction of inline leucocyte depletion, which would eliminate the (wanted) phagocytes during a period of beneficial activity (reviewed by Dzik [9]).

Also, this is naturally not regularly reported, but we have seen alterations in product quality, e.g. through red cell haemolysis by manual insufficient valve opening. Automated valve opening systems have been developed by the industry, which are hoped to result not only in a smaller amount of cell losses and benefits for working personnel, but also higher product qualities, e.g. safety to accidental haemolysis owing to insufficient manual valve breaking.

Leucodepletion

  1. Top of page
  2. Abstract
  3. Critical parameters during the donation process and in the period before separation
  4. Leucodepletion
  5. Whole blood separation and component preparation
  6. Pathogen inactivation
  7. Production of cellular blood components from undifferentiated cells in vitro
  8. Disclosures
  9. References

Leucocytes have been made responsible for side-effects of blood products, mainly in three lines: (1) febrile, non-haemolytic transfusion reactions, (2) induction of HLA alloimmunization and (3) transmission of CMV. Also, transfusion-associated Graft-versus-Host-Disease has been an issue (Müller et al., 2009 [10]). We have recently reviewed the impact of leucocyte depletion on the basis of clinical evidence (Müller et al., 2009). We identified only a few reports, allowing evidence-based conclusions on the clinical efficacy of the measure. These include data from the United Kingdom, which suggest that universal leucodepletion has further reduced the already low risk of TA-GVHD in immunocompetent recipients and has altered the profile of post-transfusion purpura cases. [11].

A further area of investigation has been potential formation of microparticles during the filtration process. Although generation of microparticles, also termed microvesicles by some authors, from red cell membranes can be seen, detrimental effects of the leucodepletion process in terms of generation of microvesicles or PrP(c) release were not observed [12]. We recently raised the question whether inflammatory mediators are still released during the production of PCs from whole blood donations, depending on the storage time (Chudziak et al., 2009 [13]). Our findings indicate that low levels of soluble potential proinflammatory mediators such as CD40Ligand can still be generated during the buffy coat preparation process, depending on the storage time. The clinical evidence of these is unclear.

Whole blood separation and component preparation

  1. Top of page
  2. Abstract
  3. Critical parameters during the donation process and in the period before separation
  4. Leucodepletion
  5. Whole blood separation and component preparation
  6. Pathogen inactivation
  7. Production of cellular blood components from undifferentiated cells in vitro
  8. Disclosures
  9. References

Separation techniques include centrifugation, buffy coat removal, manual, half automated and fully automated systems. Specific influences of single parameters will be reviewed under the specific components below.

Red cell concentrates

The possibility to store transfusable red cells relies on the presence of specific storage solutions at 4°C, which influence, among others, glycolysis, oxidative stress damage, morphology, rheological including decreased deformability, which can impede microvascular flow, and depletion of 2,3-DPG [14–17]. A clinical trial published recently in the New England Journal of Medicine has determined an adverse outcome for survival in patients who were transfused with red cell concentrates aged more than 2 weeks in patients after cardiac surgery. The results have however been seriously questioned, because for example confounding factors had not been taken into consideration that may have had a major influence on the outcome such as emergency surgery, surgical blood loss and re-exploration for bleeding, and there were clear differences in patients’ blood groups, and the proportion of leucoreduced components was significantly different between the red cell concentrates (RCC) age groups [18]. Inline leucocyte depletion has been proposed as a main factor determining immunomodulation by red cell concentrates in patients with cardiac surgery [19]. Likely, therefore, research in this field will go on with increased interest and effort in the future, with relevance for the production (and, because this is according to GMP, part of the production process) storage conditions and length of red cell concentrates.

Potentially adverse parameters during the storage of red cells include the length of storage and incidence of immunomodulatory effects [20] and description of specific parameters such as accumulation of proinflammatory mediators [21] and microvesicle formation dependent on the preseparation storage time (see above, and Ref. [22]). Vesicles were found to contain aggregated haemoglobin, band 3 and lipid raft proteins, including flotillins, Fas, procaspases and caspases cleavage products, CD47 and immunoglobulin G.

Platelet concentrates (PCs)

Platelet preparation for whole blood donations has undergone a major change with the introduction of inline leucocyte filtration and pooling on the days of production. Automated preparation systems that include the replacement of the manual pooling step have been developed, are increasingly under investigation and may serve to increase product quality, platelet yield and reliability of the process. Because few data have been published, this relevant field will however not be reviewed in more detail here.

Additive solutions

Currently, intensive work is ongoing regarding the definition of suitable additive solutions for PCs (reviewed in Ref. [23]). Major components under concern within PC additive solutions are citrate, acetate (both being made responsible for control of lactate production), and Ca, Mg (e.g. in platelet additive solution/PAS 3M; [24,25]). Extended storage is a major goal of such investigations [25]. A recent Canadian study concluded that although especially PASIII type additive solutions can yield comparable in vitro platelet function as platelets suspended in plasma, it is difficult to generate fully equivalent platelet quality [26]. Recent systematic comparisons between different storage bags or pooling systems did not result in significant differences in functional platelet parameters [27,28]. Still, work is mainly evaluated using in vitro parameters. Evaluation in vivo is difficult, and substitute parameters are scarce.

Agitation

The BEST group have evaluated the relevance of continuous agitation of PCs and reported that interruptions up to 24 h did not lead to detectable changes or deficits in platelets’ quality [29].

Storage lesion

The ‘storage lesion’ of human platelets has been observed upon their maintenance in either additive solution or plasma and has been defined through emergence of activation markers and function loss including development of abnormal forms, loss of discoid shape, decreased mean platelet volume, increased volume and density heterogeneity, increased release of α-granules and cytosolic proteins, increased procoagulant activity and altered glycoprotein expression [23]. As stated earlier, storage both preseparation and postseparation influences the presence of white cell fragments that may have impact on HLA alloimmunization [8].

Cold storage of platelets

It would be very beneficial if platelets could be stored at 40°C or even frozen, for both product availability and safety, e.g. prevention of the growth of accidentally contaminating bacteria. A specific glycosylation of N-acetyl-β Glucosamin residues by uridine 5-diphosphogalactose (UDP-galactose)′was identified as a method to allow normal survival of murine platelets after transfusion in mice (Hoffmeister et al., 2003). A recent report on cold storage of human platelets and their reglycosylation found however no functional restoration by this method [31].

Storage period of PCs

In a recent vote by its Advisory Committee on Blood (2008), the German authorities have reduced the storage time of PCs to 4 days instead of 5. This has been inferred from the fact that almost all lethal transfusions of bacterially contaminated PCs in Germany were caused by product stored between day 4 and 5. On the other hand, a magnitude of studies has investigated extended platelet storage (for example, Ref. [27]), which because of space restrictions cannot be reviewed here but are covered in Ohto et al. [23]. Bacterial detection has been pursued is a major means to extend shelf life of platelets [32]; this topic will be covered in a different publication in more detail within this ISBT Science series volume by Wood et al. (pp. 46–51) [33]. The main findings are that bacterial detection is beneficial but does not eliminate the risk of bacterial contamination [34–37]. Bacterial sampling according to GMP guidelines is considered a part of the manufacturing process.

Therapeutic plasma

Plasma preparation has a number of critical parameters, which mainly deal with the storage prior to freezing and the freezing process itself. Whereas, with regard to the latter, no considerable research activities have been recorded in the last years, storage mainly of the whole blood and its impact on plasma quality have been investigated and are reviewed in the above section on preseparation quality.

However, plasma has been recognized the major source of transfusion-related acute lung injury, mediated by plasma components from female donors with antibodies against white-blood-cell antigens being responsible for the majority of probable TRALI fatalities in a study be the American Red Cross (Eder et al., 2007 [38]). Counteractive measures have been taken that affect production logistics of plasma. This has led to the exclusion of male plasma at all in several countries or the omission of plasma from women who report having had a pregnancy during the donor interview. The logistics have affected production of therapeutic plasma.

In a preparative way, novel options of plasma usage have been proposed, e.g. by Greinacher et al., by proteomic characterization of freeze-dried human plasma and link to functionality of essential constituents of therapeutic plasma [39]. Freeze-dried plasma has been pursued; however, clinical data on the efficacy of such a product are scarce or not available at all. It is therefore questionable how without such clinical data a new blood product such as freeze-dried plasma components may be placed on the market without such information and also considering blood components licensing in the same way as for other pharmaceutical drug. Generally, however, and valid for all components, the outcome of proteomic investigations to identify causes of storage lesions can be expected to have a tremendous impact on the development of high-quality blood components.

Pathogen inactivation

  1. Top of page
  2. Abstract
  3. Critical parameters during the donation process and in the period before separation
  4. Leucodepletion
  5. Whole blood separation and component preparation
  6. Pathogen inactivation
  7. Production of cellular blood components from undifferentiated cells in vitro
  8. Disclosures
  9. References

Methods for pathogen inactivation include techniques targeting nucleic acids (for plasma as well as the cellular components) as well as techniques that target membranes that have been developed and introduced into practice for acellular products [40]. Inactivation and reduction efficiencies are high and include bacteria, parasites, viruses and leucocytes. Because there are other specific sessions and chapters on this technology and owing to space restrictions, a detailed overview cannot be given. Table 2 lists the most common methodologies that are currently in use or under development. A relevant development would be the possibility to efficiently inactivate or pathogen-reduce whole blood.

Table 2.   Overview over pathogen inactivation techniques for blood components (adapted from Ref. [40])
 Cell-free productsCellular products
Available inactivation techniquesPlasma, plasma derivativesPlateletsRed cells
Solvent–detergentYesNoNo
Methylene BlueYesNoNo
PsoralensYesYesNo
RiboflavinYesYes?
FRALE (S303)??Yes
Inactine??Yes

Production of cellular blood components from undifferentiated cells in vitro

  1. Top of page
  2. Abstract
  3. Critical parameters during the donation process and in the period before separation
  4. Leucodepletion
  5. Whole blood separation and component preparation
  6. Pathogen inactivation
  7. Production of cellular blood components from undifferentiated cells in vitro
  8. Disclosures
  9. References

The revolutionary idea to produce mature blood cells from a limited repository of somatic (or even embryonic, or reprogrammed somatic) stem or progenitor cells has in the last years been investigated with increasing intensity. Although it up to now seems very complicated to generate fully functional blood plasma in vitro (e.g. form cultured hepatocytes), individual plasma proteins are routinely produced using recombinant DNA technology, such as blood clotting factors. Ex vivo expansion and maturation from haematopoietic progenitors to red cells and platelets have been further developed, starting from cytokine-supplemented cultures of enriched progenitor cells from human cord blood towards execution of a phase-I clinical trial [41–43]. The question of the efficiency of enucleation, which has been a major concern in the beginning, is beginning to be resolved (Mirahada et al., 2006). The topic has recently been reviewed by Nakamura et al. (2009) [44].

Also, platelet generation from progenitors has been developed by several groups and includes as individual steps progenitor expansion, megakaryocyte expansion and megakaryocyte maturation [45]. Platelet-like particles have been found to lack expression of relevant platelet antigens such as CD41a and 42a (Reems et al., 2010 [46]). Instead, generated particles in some protocols, including three-dimensional scaffolds, are larger than mature platelets and are heterogeneous in size [47,48]. Possibly, many of the particles do not represent platelets or any functional analogues [49]. Major questions constitute the ideal source of the progenitors and the total amplification factor, because platelets will have to be provided at a scale of 1012 to 1013 in such a factory (Reems et al. 2010).

References

  1. Top of page
  2. Abstract
  3. Critical parameters during the donation process and in the period before separation
  4. Leucodepletion
  5. Whole blood separation and component preparation
  6. Pathogen inactivation
  7. Production of cellular blood components from undifferentiated cells in vitro
  8. Disclosures
  9. References