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Keywords:

  • blood component production;
  • platelet concentrates;
  • red blood cell concentrates

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Production methods
  5. Assessment of component quality
  6. Current challenges and limitations
  7. Future considerations
  8. Disclosures
  9. References

Continuous improvement in the area of blood component production has brought an increased emphasis on process control hand in hand with the creation of processes that provide a greater degree of standardization of the products. This work has included the systematic characterization of products produced using routine high throughput systems to understand which elements of the production process give rise either to non-conforming products or to high levels of variation in products. Such information leads to an opportunity to rethink our general approach to the assessment of component quality. Much of the recent progress in blood component production, including new developments in automation, has raised expectations of parallel opportunities to improve product quality through improved technology. The areas where we still lack a good understanding of the factors contributing to overall component quality are excellent targets for future research effort.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Production methods
  5. Assessment of component quality
  6. Current challenges and limitations
  7. Future considerations
  8. Disclosures
  9. References

The processes for production of a full range of components from whole blood donations have been available to transfusion medicine for over 50 years. The development of containers that allowed the storage of platelet concentrates was key to the establishment of transfusion therapy practices that are based on a full range of specific components rather than the transfusion of only whole blood. In the decades since glass bottles were largely abandoned, the production of components has become increasing sophisticated. The purpose of this review is to discuss modern production methods, their optimal use and the gaps that are still present to ensure component quality, and finally, the areas where future research efforts could be focused.

Production methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Production methods
  5. Assessment of component quality
  6. Current challenges and limitations
  7. Future considerations
  8. Disclosures
  9. References

It is not the intention of this article to discuss all production methods in detail. For such information, the reader is guided to the excellent detailed descriptions of Hardwick [1]. All methods for the production of components from whole blood rely on the gravitational separation of cells based on their density. At the basic level, this can be achieved with little more than a centrifuge and the manual transfer of components into unique containers. This core process is used in a wide variety of jurisdictions ranging from developing countries with technologically limited blood systems to the remaining superpower. However, technological developments have also occurred that have changed the way in which whole blood is processed into components, and more are on the way. Developed countries that are dependent in large part on whole blood collections rather than apheresis processes for the preparation of blood components have embraced the use of automated equipment to assist with component production.

The two most common component production methods used worldwide for the production of red cells, plasma and platelets from whole blood are both named for the steps required to isolate platelets for platelet concentrate production: the platelet-rich plasma (PRP) method and the buffy coat method. These two methods are outlined in Fig. 1. The PRP method is the original technique used to isolate platelets from whole blood, and it relies on applying a centrifugal force to whole blood that causes the red cells and leucocytes to separate from the platelets and plasma. Platelets may be further concentrated by a second centrifugation step. Importantly, these steps create components that are highly enriched for a particular compartment of blood but are not pure. Post-preparation steps such as leucoreduction may be applied to further enrich specific components.

image

Figure 1.  Schematic of component processing from whole blood by semi-automated production methods. A typical PRP platelet production scheme is shown in panel a, while the buffy coat production method is indicated in panel b. Both illustrate the ultimate preparation of a platelet concentrate (PC). The figure is reproduced from [7] with permission of the publisher. ©2010 Elsevier.

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Many years after the development of the PRP method, investigators in the Netherlands and Sweden developed a new method to create a platelet component. This approach was based on the isolation of the buffy coat layer from whole blood that was subjected to sufficient centrifugal force to pellet all cells away from the plasma. Under these conditions, a buffy coat layer forms on top of the red cell layer that is highly enriched in leucocytes and platelets. Several buffy coats may be pooled together, and the platelets separated from the red cells and leucocytes by centrifugation similar to the initial step of the PRP method. The drivers for development of the buffy coat method were the removal of a large proportion of leucocytes from the red cells without filtration, and the improved yield of plasma from the whole blood donation produced by the higher g forces used and the resultant tighter packing of the red blood cells.

The subsequent improvements in whole blood processing were driven by the development of equipment that facilitated the separation of components in the buffy coat process using semi-automated extraction systems. This technology is used hand in hand with the buffy coat production method, even if platelet production is not being applied to all whole blood donations. Although this instrumentation can also bring additional process control and standardization to the production of components in a PRP-based production environment, there has been surprisingly little implementation of semi-automated production aids in this setting.

An important improvement in the preparation and storage of cell concentrates has been the development of additive solutions which can either improve the product quality during storage in the case of red cell additives or, in the case of platelets, minimize the amount of plasma protein carried over to the platelet product. The use of platelet additive solutions reduces some risk of TRALI and obviates the need to test for high-titre red cell antibodies when transfusing across ABO groups. Ongoing research into the improvement of storage solutions may offer a means to minimize the development of cellular storage lesions.

The most recent developments are a natural progression from partial automation of production processes to complete automation. Initially, instrumentation was developed that automated certain aspects of the production process; for example, platelet pooling. Fully automated systems are becoming available in the marketplace, and others are under development. The challenge for this kind of new technology is always the cost-benefit analysis for a given blood system or blood centre as well as the reliance on a single vendor for a core critical supply if the disposables are tied to the automated device.

Technical improvements have also been made for frozen cellular components with the development of closed systems that facilitate the maintenance of sterility during washing steps for cryopreserved red blood cells. Frozen platelet products are also of relevance to any discussion of new developments in component production, although their use is principally restricted to the military theatre at this time.

Perhaps, the most significant change to come to component production in decades is the ability to pathogen inactivate fresh blood products. Although at this point, the technology has only been commercialized for plasma and platelets, it is only a matter of time before pathogen-inactivation techniques are also available for red cell concentrates. These technologies change the workflow and add complexity to the processes of the production laboratory, while at the same time offering opportunities to rethink our approach to blood safety.

Assessment of component quality

  1. Top of page
  2. Abstract
  3. Introduction
  4. Production methods
  5. Assessment of component quality
  6. Current challenges and limitations
  7. Future considerations
  8. Disclosures
  9. References

The treating physician must be able to use a blood component with some degree of faith that the component has been properly prepared and contains an appropriate level of functional red cells, platelets or proteins. To provide this assurance, blood components are subject to various forms of quality assurance testing. Compared to other types of manufacturing, blood component manufacture presents unique challenges, particularly with respect to the high degree of variation in the starting material. The donor’s platelet count, haemoglobin level and baseline coagulation factor activity can vary over a wide range. While we accept this as natural biological variation, it presents unique challenges in creating a set of rigorous definitions of acceptable products made in component manufacture.

With respect to quality assurance, the first step in the manufacture of blood components must be a demonstration of process control. This is assured through initial validation and the subsequent step that, in blood banking, we call ‘quality control’, although again it is unlike the application of quality control in other manufacturing environments. Most commonly, standards and guidelines recommend the testing (usually destructively) of a minimum number of products or 1% of the manufacturing run over some unit of time (commonly per month). This approach is somewhat empirical and presumes that there is little variation from run to run. It is also inadequate to quickly identify relatively rare but important negative events such as sterility breaches as these can occur in < 1% of products and are thus difficult to find using this kind of process control practice. Increased focus is coming to other methods of demonstrating that a process is in control notably the use of statistical process control tools which bring increased rigour to this type of quality assurance [2]. Statistical process control is more appropriate to use for determination of process control when one has a process that is inclined to be at greater risk of being out of control; in process, leucoreduction by filtration is an example of such a process. Importantly, quality control and process control data can be seen as an early warning system that must be regularly interrogated to determine whether unusual trends are developing. Even if processes and systems are demonstrably in control and regulatory authorities are satisfied, critical inspection of these data on an ongoing basis will often catch problems before they get out of hand or have clear impact on product quality. For example, small but unexpected fluctuations within a data set for the haemolysis levels of red blood cells at outdate may indicate that it is time to initiate staff refresher training in the production laboratory [3]. Another example, shown in Fig. 2, shows data drift over time in residual white cell levels in leucoreduced products. Timely intervention returned the quality control values to the usual range and prevented ongoing QC failures.

image

Figure 2.  Assessment of residual leucocytes in leucoreduced PRP platelet concentrates. Data points represent the sequential measurement of leucocytes in quality control of 1% of manufactured inventory. The data indicate an increasing loss of control of the process that eventually led to failure to meet the standard. Intervention was made at the point indicated by the arrow, and test values rapidly fell to earlier levels. Data analysis courtesy of Craig Jenkins, Canadian Blood Services.

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In addition to a requirement to demonstrate that the production process is in control, there is a need to be able to assure that all blood products are of appropriate quality. This is an area in which blood component manufacture differs in some important aspects from the usual understanding of quality indicators. The quality control standards that are applied to components are, for the most part, surrogate measures of product quality in that the measure may only be altered when the product quality is markedly deficient. For example, the pH of a unit of platelets may not drop below the minimum standard, but the platelets contained in that unit may be of poorer quality than one would expect because of variation in the effect of storage time on platelets. More direct measures of product quality are normally made only in special circumstances as they are not mandated by regulatory authorities. A listing of potential quality measurements for platelet and red cell components is given in Table 1.

Table 1.   Commonly used quality measures for fresh cellular components
Tests for platelet quality
 Measuring platelet activation state• CD62P expression • Annexin V binding • Extent of shape change • Morphology score • Swirl
 Measuring platelet metabolic activity• Glucose • Lactate • pH
Tests of red cell concentrate quality
 Measuring red cell metabolism• 2,3- DPG • ATP and other nucleotides • Glucose
 Measuring red cell integrity• Osmotic fragility • Potassium • Morphology • CD47 level • Annexin V binding • Per cent haemolysis at outdate

Users of blood transfusion products are questioning with increasing frequency the quality of the products that we manufacture. Notably, recent discussion has focused on the clinical significance of the age of red blood cells, the age of platelet products and the effects of pathogen-inactivation techniques on the quality of plasma and platelet components. One area in which we have changed quality control practices in many blood systems is around the use of bacterial testing of platelet products. Although it is not normally so-named, this is a type of quality control test and is one of the few tests that we may perform on every unit of product depending on the jurisdiction.

The use of our products is also becoming more sophisticated with considerations being given to transfusion of red blood cells based on donor blood volume and haemoglobin concentration in the unit [4]. Similarly, the appropriate dose of platelets is also a focus with recent studies being conducted of a spectrum of platelet doses [5,6]. These studies have been unable to take variations in product efficacy into account. The ‘effective dose’ of a transfusion product becomes much more important in such settings. Although most of this discussion has focused on fresh cellular components, one should not lose sight of the need to make relevant efficacy measurements in transfusion plasma as well. Our existing quality standards were designed at least in part to have the ability to monitor FVIII for fractionation as part of the raw material qualification. The characteristics of transfusion plasma that are the most important to ensure efficacy have yet to be clearly delineated, in part perhaps because of the lack of clarity around the use of plasma for transfusion in many hospital settings.

Storage lesions occur in both red blood cell and platelet concentrates. It is useful to monitor some aspects of storage lesion development as part of quality assurance, but there is no agreement as to which measures are the most appropriate or when to measure them. For platelet concentrates, most jurisdictions require only pH, platelet count and minimal sterility testing as quality standards, yet there are clearly other possible tests to perform. Some consensus should be sought within our field as to appropriate additional measures to assess the development of both the platelet and red cell storage lesions.

Current challenges and limitations

  1. Top of page
  2. Abstract
  3. Introduction
  4. Production methods
  5. Assessment of component quality
  6. Current challenges and limitations
  7. Future considerations
  8. Disclosures
  9. References

There are many interesting challenges facing component production. They range from questions of how to optimize the collection and storage of blood (do blood bags have to be flat, floppy and awkward to store?) to how to ensure product efficacy. Our standards for blood products were developed at a time when our ability to measure meaningful characteristics of blood components was limited by both our technology and by our scientific understanding of the cells and proteins themselves. Several decades later, it is time to look hard at modernizing the standards to which we measure components so that they reflect better the efficacy of the transfusion products we give to patients.

Blood component production has become increasingly aligned with the practices in the pharmaceutical industry for the preparation of biological drugs. Optimally, components are produced in laboratories that adhere to good manufacturing practices (GMP) using raw materials that were collected using similar GMP philosophy. However, blood component production faces some unique challenges compared to most drug-manufacturing environments. First of all, there is a very high degree of variability in the raw material; in fact, each donor might be considered to be their own ‘lot’. The blood centre is used to accepting anyone who is willing to donate and who passes the donor screening process. However, it may be worth considering that applying somewhat more sophisticated ‘raw material’ handling practices could increase the quality and consistency of the products that we produce. Just as we have made such changes to optimize the relationship between inventory and demand, we should carefully consider novel ways to approach the relationship between donor characteristics and product characteristics.

A better understanding of optimal quality parameters will become increasingly important as we move to the next set of challenges facing component production produced by the application of pathogen-inactivation technologies. This is a breakthrough technological improvement in blood safety, but it comes with a price paid in a reduction in component quality. We must gain better tools with which to assess pathogen-inactivated products to align with the widespread implementation of this new step in the component production process.

Future considerations

  1. Top of page
  2. Abstract
  3. Introduction
  4. Production methods
  5. Assessment of component quality
  6. Current challenges and limitations
  7. Future considerations
  8. Disclosures
  9. References

Many of the issues that the component production area face are actively being addressed, but many remain to be tackled. For some of these, it is clear that additional scientific and engineering progress will be required before solutions will be at hand. While every person who prepares or uses blood components will have their own list of things that they wish could be improved, the following section identifies just a few areas that require the additional effort and focus of the transfusion research community, both in the public and private sectors.

Quality control needs to be made more meaningful. We must identify measurements that can be made simply and inexpensively before the product is released to the hospital that will accurately predict product quality. Those measurements that we make should be more clearly predictive of product efficacy. In the absence of successful blood substitutes, the manufacture of blood components will always suffer from a high level of variation in its raw material, the donor. What real time information can we provide that is specific to each product that will ensure optimal use of the product with respect to dosing and efficacy?

Ideally, we need to make components in the most consistent manner possible by paying careful attention to each part of the production process from donor selection to component production to product assessment. We need to conduct the research required to modernize our standards such that we identify parameters that can be measured in non-destructive testing systems using products that are still within their manufacturing window. The development of quality measures will work in an iterative process with the development of improved production techniques to continue the modernization of blood component preparation for the benefit of patients.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Production methods
  5. Assessment of component quality
  6. Current challenges and limitations
  7. Future considerations
  8. Disclosures
  9. References