Blood processing

Abstract

A unit of whole blood collected from a donor is a precious asset. Gone are the days when the blood collected was simply stored, and then transfused to a patient as whole blood. A donation of blood should be seen as a bag containing all the different constituents of whole blood – red cells, white cells, platelets, and plasma with plasma proteins such as clotting factors and protective antibodies. In modern transfusion medicine the aim is to transfuse the patient with only the component required. For example, if a patient is deficient in red cells only, then why transfuse the other components that are not required? They could be better utilized for patients who lack those specific components. Whole blood may be processed into various components. Each component can then be stored under ideal storage conditions (i.e. temperature and movement) to ensure that the product is most effective when it is used. Special preservative solutions and blood bags are used to lengthen the expiry time and improve product quality. ‘Component therapy’ also maximizes the use of one donation. The products from a single donation can benefit multiple patients. In this way the best use is made of a scarce resource as well as conforming to sound economic principles. This section will cover very broadly, the processing of blood into components. By the end of this section the student should be able to relate the benefits of processing blood into components and to discuss the requirements of a component programme under the following headings: Concept of blood components Aims of blood transfusion Why components? Processing blood into components Sterile systems Bag systems for blood collection Single bag Double bag Triple bag Quadruple (quad) bag Top and bottom bag Principles of centrifugation Blood processing equipment Blood bag centrifuge Scale Balances Plasma extractor Automated blood processing machines Pilot tube sealer Sterile connecting device Platelet agitator Plasma snap freezer Low temperature freezer Laminar flow cabinet Preparation of specific blood components Whole blood Red cell concentrate red cell concentrate in plasma red cell concentrate, buffy coat removed, in additive solution (red cell concentrate, leucocyte-reduced) red cell concentrate, filtered (red cell concentrate, leucocyte-depleted) prestorage on demand in line add on red cell concentrate, paediatric red cell concentrate, cryopreserved (frozen) red cell concentrate, washed Plasma general information fresh frozen plasma fresh frozen plasma – for fractionation freeze-dried plasma cryoprecipitate Platelet concentrate general information preparation of platelet concentrate by platelet-rich plasma method preparation of platelet concentrate by buffy coat method conventional quad bag method top and bottom bag method single unit platelet concentrate from buffy coat pooled platelet concentrate from buffy coat pooling kits chain method apheresis platelets quality control for platelet concentrates Irradiated products Autologous preoperative donations Measures to prevent transmission of pathogens Labelling and records Quality control Overview of plasma fractionation. Blood is usually transfused to provide the means of maintaining oxygenation of the tissues, or to correct bleeding and coagulation disorders. The role of each blood component, when transfused, is briefly as follows: Red cells (erythrocytes) maintain oxygen supply to the tissues when patient's red cells are lost as a result of bleeding or as a result of anaemia. Platelets (thrombocytes) correct bleeding disorders caused by thrombocytopaenia. Plasma is sometimes given to maintain blood volume after haemorrhage resulting from trauma or surgery. fresh plasma may be used to replace clotting factors and assist with the correction of abnormal bleeding. plasma may be fractionated to manufacture derivatives to treat clotting factor disorders, to provide short term immunity with immunoglobulins, or to treat burns with albumin. White cells (leucocytes, including granulocytes) are not routinely transfused. They may contain transfusion transmissible infections (TTIs) such as CMV and on the other hand cause immune modulation, both of which would be detrimental to the patient. Although there may be some indications for granulocyte transfusions, in broad terms, component therapy aims to reduce the number of white cells transfused. It is clear that blood has many functions. If a whole blood unit is processed into specific red cell, plasma and platelet components there are clear advantages for the patient and for the blood transfusion service: A single donation of blood can benefit several different patients. This is the rational use of a scarce resource. Patients receive only the component(s) necessary to treat their condition. Components that the patient does not require are not transfused (such as white cells or plasma proteins when the patient requires only red cells). This reduces the risk of transfusion reactions. The storage conditions of the products can be optimized by the correct choice of additive, temperature, bag type and other parameters to ensure effectiveness of each component for the longest possible time. Figure 11·1 shows the three main component types (red cells, plasma, platelets) that can be processed from blood and lists some of the products that can be made from them. Components from whole blood. To process whole blood into components requires a basic understanding of: Sterile systems Blood bag systems Principles of centrifugation Blood processing equipment Preparation of specific blood components. Blood bag manufacturers must ensure that all blood bags plus anticoagulants and additive solutions are sterile (free of contamination by bacteria or viruses) and pyrogen free (do not contain endotoxins or micro-organism debris). At the time of blood donation, the venepuncture site on the donor's arm is thoroughly cleaned and the sterile needle on the end of the blood bag tubing is inserted into the vein. In this way a direct connection is made between the bloodstream of the donor and the sterile blood bag. At the end of the donation the tubing is sealed, and the contents of the blood bag should be free of any bacterial contamination. Once the blood bag has been sealed it is considered to be a ‘closed system’ and this is most desirable as the integrity of the product is ensured. If at any stage after being sealed, the bag is either deliberately or accidentally opened, it is considered to be an ‘open system’ (i.e. environmental air, which contains microbial aerosols, could have entered the bag). Accidental opening of a blood bag means that the blood bag must be discarded as sterility can no longer be guaranteed. This is because micro-organisms are able to gain access to any product that has been opened to the environment. Sometimes a blood bag is deliberately opened under controlled conditions (e.g. using a laminar flow cabinet) to make a specific blood product. In these cases a shortened expiry time, usually up to 24 hours from time of opening, is applied to the product. The routine processing of blood into components relies on the availability of closed blood bag systems. Individual bags in multiple bag systems are connected with one another via tubing and thus constitute a closed system. The advantage is that the components can be squeezed into the attached bags (after centrifugation) without affecting sterility, as the primary bag is never opened and the entire process occurs in a closed system. A wide variety of polyvinyl chloride (PVC) plastic blood bag systems is available from many suppliers. The choice of bag depends on the requirements of the particular blood service: Affordability Clinical demand for components (such as red cell concentrates, plasma, platelet concentrates) Whether units will be processed manually or automatically Level of product storage to be achieved (additive solutions, special plastic bags for platelets) Whether filtration (leucodepletion) is required. The following blood bag descriptions provide an overview of some available configurations. Some examples of the situations in which the configuration could be used are also given. Diagrams used to illustrate bag systems throughout this section are not necessarily drawn to scale. This is the simplest of bags available. The donation is taken into the bag and the pilot tubing is then sealed. No further processing into components is performed and the unit is transfused as whole blood. The bag contains an anticoagulant solution (CPDA). Typically this contains sodium citrate that prevents clotting, and citric acid (C), monobasic sodium phosphate (P), dextrose (D), and adenine (A) that provide buffers and nutrients for enhanced red cell survival. In a multiple bag system the bag with anticoagulant into which the donation is taken is referred to as the primary bag. This is exactly the same type of primary bag as the single unit, but in the double bag system an additional empty bag is attached (called a transfer or satellite bag). After centrifugation of the whole blood, the plasma can be transferred through the tubing to the attached transfer bag creating two components, a red cell concentrate suspended in plasma (in the primary bag) and plasma (in the transfer bag). Figure 11·2 is a diagram of a double (two bag) system. Diagram of double bag (two bag system). A triple bag differs from a double bag only by having an additional transfer bag. Once plasma is separated into the first transfer bag, there is still another empty transfer bag attached to it. This configuration is used to manufacture platelet concentrates from platelet rich plasma, or to harvest cryoprecipitate from fresh frozen plasma. See detail later in the section, under Preparation of specific blood components. Figure 11·3 is a diagram of a triple (three bag) system. Diagram of triple bag (three bag system). A quad bag system is similar to the triple bag system but has an additional bag containing red cell additive solution, and is usually used in automated systems to prepare: Red cell concentrate (RCC), from which the white cells have been removed, and to which additive solution has been added. The benefits of this are discussed under Preparation of specific blood components. A bag containing the buffy coat (BC) layer of white cells (leucocytes) and platelets which forms at the interface between red cells and plasma after centrifugation of whole blood. This layer is squeezed from the primary bag into one of the transfer bags and can be discarded or used for the production of platelet concentrates. Details of this process are described under ‘Preparation of specific blood components’. A unit of plasma that is squeezed from the primary bag to one of the transfer bags. Figure 11·4 shows a quad bag (four bag system). Diagram of quad bag (four bag system). This is a special three bag system designed for use in automated systems. The primary collection bag has an empty transfer bag connected to the top, and another transfer bag containing additive solution connected to the bottom. After centrifugation the bag is placed in an appropriate blood processing machine that expresses the plasma out of the top and the red cells out of the bottom leaving the BC in the primary collection bag. The same three products obtained in the quad bag system are created: Red cell concentrate (with buffy coat removed and suspended in additive solution) in the bottom transfer bag. A BC layer of white cells and platelets that remains in the primary bag. This BC can be discarded or used for platelet concentrate production. Details of this process are described under Preparation of specific blood components later in this section. A unit of plasma, which is squeezed from the primary bag to the top transfer bag. Figure 11·5 is a diagram of a top and bottom bag system. Diagram of top and bottom bag. The decision to remove BCs from RCCs or not, should be made by the management team of the transfusion service. There are advantages to removing the BC: The process makes the RCC leucocyte poor (not the same as leucocyte-depleted) and reduces micro-aggregate formation during storage. All the plasma is removed and can be stored as fresh frozen plasma and used as such or as a starting material for fractionated products. The red cells can be resuspended into a solution designed to offer optimal conditions for red cell storage, e.g. saline-adenine-glucose-mannitol (SAGM). The BCs can be used for platelet concentrate production. Creating these sophisticated products usually requires either a quad or top and bottom blood bag system and automated processing equipment. The cost of bags and machines may make it impossible to implement these processes where collection and demand are not high enough to justify the system. Simpler double or triple bag systems may be more practical in these situations. Blood constituents can be separated because they differ in size and density and will sediment at different rates when centrifugal force is applied. When whole blood, which is a mixture of cellular components suspended in plasma and anticoagulant, is centrifuged, the red cells settle at the bottom of the blood bag because they have the highest density (have a greater mass/weigh more than the other components). Being less dense, the white cells and platelets do not settle as quickly, and remain in suspension longer. As centrifugation continues, the white cells sediment above the red cells, and finally the platelets form a layer above the white cells and leave the original suspending fluid (now clear plasma plus anticoagulant) at the top. Figure 11·6 illustrates the separation of components as a result of moderate or hard centrifugation of a unit of whole blood. Centrifugation of whole blood. The choice to be made is the speed and time of centrifugation to be used in order to separate the desired component. For example, if platelet rich plasma is required then centrifugation should be stopped before platelet sedimentation begins. A lower centrifugation speed for a longer period would make it easier to select the time to stop. If, on the other hand, cell-free plasma is required then a faster centrifuge speed for an adequate time period would yield clear plasma and densely packed red cells. It is important that the optimal conditions for a good separation be carefully evaluated for each centrifuge to obtain the desired components. To establish optimal centrifugation: Collect parameters that indicate the desired outcome of the procedure [e.g. whole blood centrifugation should yield RCCs with a certain haematocrit (packed red cell volume) and a certain volume of plasma as well as a BC with a particular platelet yield]. From the centrifuge manual or other procedure manuals establish a base line centrifuge setting with regard to speed (rpm or g) and time. Prepare a number of products (more than 10) using this setting and measure all parameters. Repeat the procedure with reduced or increased speed and time combinations and compare parameters until the optimum setting for the centrifuge is established. After centrifugation, the bag system is carefully removed from the centrifuge (to prevent mixing) and the primary bag is placed in a plasma extractor, or an automated processing machine, for separation. Pressure is applied to the bag and the component layers are then transferred, in order, into one of the transfer bags connected in the closed system. Blood bag centrifuges are essential pieces of processing equipment. Several manufacturers market centrifuges capable of spinning 4–12 blood bags at speeds of up to 5000 rpm (revolutions per minute). They are large machines that are usually floor standing and require dedicated floor space and electrical supply. Professional installation and a good maintenance programme are essential to ensure staff safety and consistent reproducible centrifugation of product. A typical centrifuge has a motor that turns a rotor housed in a very thick metal chamber. As the rotor is spinning the door on this chamber seals automatically to prevent access by the operator as this would be extremely dangerous. The rotor is designed to hold a certain number of metal buckets. These vary in size and shape depending on the type of blood bag system being centrifuged (e.g. a quad bag system with additive solution will require a bigger bucket than a simple double bag). Each bucket has a plastic insert that can easily be loaded into, or unloaded from, the metal bucket. The inserts make the centrifugation process easier to perform and are easy to clean. Figure 11·7 shows a centrifuge bucket insert, metal bucket and rotor that fits into a centrifuge housing. Centrifuge insert, bucket and rotor. When loading a rotor, the bucket/insert/blood pack combinations that are placed opposite one another must be of equal weight. The metal buckets are rarely removed and should be placed in the rotor according to matching weights. Two plastic insert and blood bag combinations are placed on a balance (or scale). Weight (mass) in the form of plastic or rubber strips, is added to the lighter combination until the two bag/insert combinations are of equal mass. The loaded and balanced inserts are then placed into the metal buckets opposite one another in the rotor. Failure to balance the buckets before running, can cause the centrifuge to vibrate when turned on, and could cause serious machine damage or injury to personnel, and the product will not be adequately centrifuged. The use of water or other liquids is not recommended for balancing as the liquid may become bacterially contaminated, and moisture may smudge or dislodge the blood bag label. The plastic or rubber strips used for mass correction should be disinfected regularly. Blood pack centrifuges must also have a refrigeration capacity that enables the blood temperature to be controlled during processing. In order to make quality blood components, a centrifuge must be able to perform within tight parameters. The amount of ‘spin’ (centrifugation) that a product requires can be measured in terms of speed and time (e.g. 2000 rpm for 10 minutes). This, however, is not the most accurate way, as the amount of gravitational force exerted is much harder (more intense) in centrifuge heads with a longer radius (2000 rpm in a centrifuge with a radius of 30 cm is a much softer (less intense) spin than 2000 rpm in one with a 50 cm radius). It is therefore better to calculate the g force for a particular spin, and specify the requirements in terms of gravitational force and time (e.g. 1900 g for 10 minutes). This figure takes the centrifuge radius into account and can be used to set similar centrifuge settings on different makes and models. The handbook related to the blood pack centrifuge provides the formula to convert rpm to gravitational force for the machine. Some will have built-in software to do the conversion automatically. Sophisticated centrifuges also take into account the time taken to reach the desired speed (acceleration) and the time taken to stop (deceleration/braking), as these will vary according to centrifuge load. It is also possible to link computer software to a blood pack centrifuge and record all ‘run’ (operational) data for total process control. This means that the software is able to capture information like operator's name, date and time processed, and details of the centrifuge time and speed parameters against the donation identification number. In the event of quality problems, full traceability of the centrifugation data is thus available. A laboratory scale capable of weighing components to at least the nearest gram in mass (weight) is essential when making components. The mass forms a critical part of quality control, for example: Whole blood must meet mass requirements in order to be suitable for component production. Each component produced must fall within a specified mass range. The mass may be used to calculate the volume of a component (gross weight of component minus empty bag weight, multiplied by specific gravity of component). Systems need to be in place to ensure the accuracy of the scale on an ongoing basis and a calibration/service should be performed at least once per year. Figure 11·8 shows a laboratory scale for weighing blood components. Laboratory scale for weighing components. A balance has two weighing platforms and is used to prepare combinations of blood bags/centrifuge inserts of equal mass to be placed opposite one another in a centrifuge. The balance should ensure that combinations do not vary by more than 1 g. Clean plastic or rubber pieces are added to the side with the lighter combination until it is equal in mass to the heavier side. The display will indicate the mass difference in grams and give the operator a guideline as to how much balance material to add. A calibration/service should be performed at least once per year. Figure 11·9 shows an example of a balance with two weighing platforms for preparing blood bags of equal mass for centrifugation. Balance with two weighing platforms for preparing blood bags for centrifugation. A plasma extractor (or blood press) is a commercially available device that is used to apply pressure to a centrifuged unit of blood in order to transfer part of it (e.g. plasma, BC) to an attached transfer bag. The design of the device is such that a controlled amount of pressure is applied to the bag that should allow reasonable flow of liquid from one bag to the next without danger of bursting the bag or causing excessive frothing of the component being transferred. Figure 11·10 is a sketch of a plasma extractor with the handle locked open for placement of the blood bag. Plasma extractor. Regular cleaning and checking of the device is essential and if it is not performing correctly it should be repaired before use. Additional pressure should not be applied by squeezing the plates together by hand to ‘speed things up’ or compensate for a lack of pressure as a result of a defect. To operate the machine, the following steps are taken: Use the handle to open the front pressure plate. Hold it in the open position using the hook provided. Carefully remove the centrifuged blood bag from the centrifuge bucket. Hang the primary bag on the hooks located on the backing plate. Great care must be taken not to disturb the interface between red cells and plasma. Carefully reposition the transfer bags still attached to the primary bag and place them on the workbench next to the plasma extractor. Release the handle and slowly allow the front pressure plate to apply pressure to the primary bag in the press (without disturbing the interface). Once the pressure is applied, break the seal on top of the blood bag to allow plasma, and later BC if desired, to flow via the connecting tubing into the transfer bag. (Do not release pressure in mid-flow as this will cause mixing in the bag.) Stop the flow at the desired point by using forceps or plastic tubing clamps to create a temporary seal in the tubing. Now remove the separated bags of components from the extractor. Permanently seal the blood pack tubing for the separation of the bags into individual components. Automated blood processing machines are commercially available devices that can be configured to process a centrifuged unit of blood into the required components with little or no manipulation by the operator other than to load and unload the machine. There are several different types available that vary from machines that perform basic separations to more sophisticated machines that perform advanced separations and record the details of each separation for total quality management purposes. The machines use light sensors to detect blood cells in the primary bag and tubing to activate the selected programming that controls the opening and closing of tubing clamps and regulates flow between bags. Some machines also have built in scales that weigh the product transferred to the bags (e.g. BC) and use this information to activate the clamps. Fully automated blood processing machines will also perform the sealing of tubing between bags as part of their process. Automation is justified when: There is an adequate number of units requiring processing each day. The transfusion service has made a decision to produce leucocyte-reduced red cell concentrates (i.e. BC removed) that are suspended in red cell additive solution. The transfusion service wants to produce platelet concentrates from BCs. Personnel are motivated by the idea of automation and the quality improvement that can be realized by moving to it. Adequate technical support is available in the area for the repair, maintenance and calibration of the machines. The top and bottom bag is an ingenious three bag system designed specifically for use with blood processing machines. Other quad bag systems with additive solution attached are also designed specifically for automation. The manual processing of units of whole blood into red cell concentrates suspended in additive solution, involving the removal of BC and plasma into separate transfer bags, is an extremely labour intensive operation to perform routinely without the aid of automation. After manual and semi-automated processing the tubing between separated components is temporarily sealed using forceps or plastic clamps. These temporary seals are replaced with permanent seals in the tubing as soon as possible. Seals are also used in the tubing of prepared red cell products to make segments of approximately 5 cm in length, that contain red cells from the product for use in testing without compromising the sterility of the bag contents. A tubing sealer is a fairly simple device that, by means of heat, creates permanent seals in the PVC tubing of blood packs. The ideal seal is made quickly without generation of excess heat and will be about 2 mm wide with a ‘split line’ down the middle to enable easy parting of the tubing when firmly pulled apart. Figure 11·11 shows an example of a pilot tube sealer. Pilot tube sealer. There are several types available: Hand held types that are either electric or battery powered and are used at the bedside and in operations where the sealer is brought to the bag. Bench model types that are positioned in specific areas where the bags are brought for sealing and are used when a large number of seals need to be made. They are generally quicker and less prone to overheating than the hand held machines. Great care needs to be taken to ensure that the machines are functioning correctly. Without a proper cleaning, maintenance and quality checking system, products with faulty or leaking seals might get into the blood supply. A sterile connecting device (SCD) is used to attach an additional transfer bag (or bags) to a primary blood bag without breaking the sterile integrity of the system. The shelf life of components thus prepared is the same as if the product had been prepared in a closed system. The pilot tubing of the primary bag is placed into a slot on the SCD. The tubing of the transfer bag to be joined is placed in another slot running parallel to the first. On starting the operation a disposable wafer is superheated by the machine and then drawn through the tubing in the slots. Simultaneously, the tubing is moved to align the ends to be sealed, and the wafer is withdrawn. The ends to be sealed are welded together instantly and the closed system is extended by another bag (or set of bags). Figure 11·12 shows a sterile connecting device and illustrates how the tubing of two separate bags is joined to extend the closed system. Sterile connecting device. The ability to extend a closed system has many advantages as it allows the technologist to weld in bags with filters, smaller bags to create paediatric units, and to create platelet pools. All SCD welds must be inspected for quality, integrity, leaks, air bubbles and alignment. The consequences of passing a faulty weld can be very serious indeed, so procedures must be in place to ensure that the correct course of action is taken when faults are detected. Record keeping must include full documentation of products welded and weld quality control results. Regular (once per year minimum) servicing and calibration of weld strength is vital to safe use of the SCD. A platelet agitator is a device designed to fulfil the need for platelet concentrates to be agitated during storage and is an essential part of the equipment in a modern components laboratory. The best mixing action is provided by a machine that moves a flat tray/shelf in a gentle horizontally oscillating motion (side to side) at approximately 70 cycles per minute. Other types, which rotate end over end, or in an elliptical action, are considered to have too robust an action and are not ideal. Figure 11·13 is a sketch of a small platelet agitator. Platelet agitator. The oscillating tray/shelf is made of mesh or stainless steel sheet with multiple holes punched through it so that when a platelet concentrate bag is placed on it, air can circulate all around the bag. This helps to fulfil the need for platelets to exchange gas through the walls of the special bags used for platelet storage. Another requirement of platelet concentrate storage is that they be held at controlled room temperature (+22°C ± 2°C). Platelet agitators that are built into a temperature controlled cabinet are ideal, but if the agitator is operated in a room where a controlled room temperature environment is maintained (and recorded) they are not essential. See Platelet concentrate, general information later in this section for more on platelet storage requirements. A strict cleaning routine according to the manufacturer's specifications should be in place to ensure that the entire machine (in particular the oscillating shelves) is clean and free of bacterial growth. Records indicating the fr