Blood Component Processing and Storage

Chapter 17
Blood Component Processing and Storage

Cheryl L. Mansell¹ and Manuel Boller²

¹Australian Red Cross Blood Service, Melbourne, Victoria, Australia

²U-Vet Werribee Animal Hospital, Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Werribee, Victoria, Australia

Introduction

The process of converting a unit of whole blood (WB) into blood components is an achievable outcome for many veterinary practices. The practice of component therapy in veterinary transfusion medicine is, to a large extent, extracted from human data, as is the method and background of blood component processing and storage. Aside from the resources needed for collecting WB, the materials required for blood component production include blood collection containers or packs and processing equipment. Commercially available blood collection packs are sterile and exist in a variety of configurations to facilitate the production and storage of specific blood components. The anticoagulants and red cell additive solutions can differ and can impact the shelf-life of blood products. Some blood packs will also include in-line leukoreduction filters. Thus, the choice of appropriate materials is an essential step when planning the manufacture of blood products. Moreover, the appropriate types and use of equipment, including centrifuges, refrigerators, and freezers, is essential for the production and storage of high-quality blood products. This chapter describes the techniques and methods for processing WB collections into components to help ensure the product is suitable for storage and transfusion, thus minimizing the risk to recipients. Quality control and quality assurance of the blood product and equipment are essential parts of blood processing and are also described in this chapter. Note that while apheresis can be used to separate components at the point of bleeding, this is not a realistic process in most veterinary practices, therefore the focus of this chapter is component separation of WB.

Processing

Processing supplies and equipment

The process of converting WB into components requires little in terms of specialized equipment (Box 1). Many items can be obtained second-hand from human blood banks as they regularly update their equipment.

Whole blood collection pack

The configuration of a blood collection set influences what blood components can be prepared. The common style of blood collection set is a triple pack, but single, double, and quadruple packs are also available for canine WB collection. Small volume blood packs suitable for closed-system blood collection and processing in cats are also available. A commercially prepared blood collection pack is sterile and will generally consist of a primary collecting bag containing anticoagulant with satellite bags. Such a sterile pack is termed a closed-collection system as it permits bag-to-bag transfer of blood or its components without exposure to the environment. A triple pack allows the production of packed red blood cells (PRBCs) and plasma, the most common blood products used in veterinary practices. Plastic characteristics used to manufacture individual bags within a pack can vary to fulfil the requirements of each bag’s specific purpose. For example, the plastic PL146 is suitable for freezing of plasma products but not suitable for platelet storage as it does not permit adequate gas exchange, required by functioning platelets (Prowse et al. 2014). Therefore, these blood packs should only be used for the intended products as per the manufacturer. Bags have additional features designed to make processing easier, for example a pair of holes (eyelets) at the top of the primary collection bag (Figure 17.14a, arrows) to facilitate positioning on the plasma extractor.

c17f014 — **Figure 17.14** a. Set up for plasma expression in plasma press. Note the use of the bag eyelets for vertical stable positioning of the bag in the plasma press (arrows). b. Spring plate is unlatched to apply pressure to blood donation. When connector seal (black arrow) is broken, plasma is allowed to flow into satellite bag. c. Spring plate is latched to remove pressure from blood donation at the end of the plasma separation process.

Box 17.1 Supplies and equipment essential for blood processing

Fresh whole blood collection pack
Scales (Figure 17.1)
Centrifuge – refrigerated (Figure 17.2)
Plasma press (Figure 17.3)
Hand sealer or line sealer with or without clips (Figures 17.4–17.7)
Donor tube stripper/line stripper (Figure 17.8)
Scissors
Atraumatic clamps (Figure 17.9)

Figure 17.1 Digital scale.

Figure 17.2 Floor centrifuge.

Figure 17.3 a. Manual plasma press. b. Automated plasma press.

Figure 17.4 Line sealing, using two metal clips (bottom) or heat seals (top).

Figure 17.5 a. Hand sealer. b–e. Correct use and end result of line sealing with rectangular metal clips. f, g. Incomplete line sealing due to hand sealer exerting uneven pressure on the clip.

Figure 17.6 a–c. Dielectric (heat) sealing.

Figure 17.7 Handheld dielectric tube sealing device.

Figure 17.8 a. Line stripper. b. Proper functioning line stripper allows complete evacuation of fluid from tubing. After line stripper release, the tubing will immediately fill with a representative sample of blood bag content.

Figure 17.9 Tools for temporary clamping include plastic haemostat clamps (top) or plastic clips (bottom) for use during centrifugation.

Blood collection packs can be modified using a sterile connecting device or welder, which permits insertion of a leukoreduction filter or addition of extra satellite bags. The device welds two separate sterile pieces of tubing into one. If the blood collection process is validated (see the section on quality control and quality assurance), the resulting product can be regarded as a closed system (European Committee on Blood Transfusion 2013). The WB collection should be received for processing with the blood collection needle removed and the tubing sealed by either clip- or heat-sealing (see the section on tube sealers).

Scales

Scales are necessary to weigh blood products and for balancing centrifuge contents (Figure 17.1). The scales should be digital to permit recording of accurate weights, allow a minimum resolution of 1 g, and have a capacity of up to 2 kg.

Centrifuge

The centrifuge must be purpose built for the processing of donor blood in order to withstand the high centrifugal forces required to separate the blood. The centrifuges used are typically large-capacity floor models that can process two to six units of WB per centrifuge cycle (Figure 17.2). The shape of the centrifuge buckets, usually circular or oval, will depend on the brand of centrifuge. It is important that the centrifuge is refrigerated to allow thermal control during the centrifugation process; friction-related heat generation will otherwise impair the quality of blood components. Depending on the manufacturer and model, centrifuge features such as the nature of the interface, availability of acceleration and deceleration settings, presence of balancing stabilizers and imbalance detectors, and preset capability vary widely. Consultation with the manufacturer or sales representative for a detailed appraisal of the centrifuge and advice regarding set up and features is recommended.

Plasma press

A plasma press is designed to allow gentle plasma removal from PRBCs, platelets, or cryoprecipitate (CRYO) after centrifugation. If using blood collection sets intended to retain the buffy coat (BC) for platelet production, a specifically designed plasma press must be used.

Various plasma press styles are available. The simplest form consists of a spring-loaded platform that applies consistently distributed pressure on the blood pack (Figure 17.3a). This press will likely have a pair of prongs towards the top for stable vertical positioning of the blood collection unit. Since this device requires the operator to manually stop the plasma expression, the amount of plasma expressed and the extent of potential contamination of the plasma supernatant with cellular material is influenced by the skill of the operator. Semi- and fully automated plasma presses (Figure 17.3b) that complete the expressions with limited or no input from an operator are also available.

Tube sealers

A sterile point of separation between blood packs is achieved with the use of a tube sealer. The tube sealer must completely seal the tubing, with two separate seals made between the end of a line and the blood product to maintain sterility (Figure 17.4). The tubing can be sealed with the use of metal clips or dielectric sealing.

A hand sealer (Figure 17.5a) requires the use of metal clips, either rectangular or round, that are placed on folded pieces of tubing by the operator. The hand sealer is specific to the type of clips used. Some designs of hand sealers also incorporate a tube stripper. Rectangular clips are designed with an indentation on the short side, which forces the clip to crimp when compressed with the hand sealer. The clip should be checked after application to ensure it has crimped completely and evenly (Figures 17.5b–g).

Dielectric sealing utilizes a high-frequency current passing between two electrodes for precise localized heat generation. The plastic tubing is placed between the electrodes as they press together, thereby melting the plastic and forming an impenetrable seal (Figure 17.6). In addition, the sealing site centrally splits easily when applying traction, while leaving both ends of the tube occluded. This permits convenient and clean separation of tubing segments. Dielectric tube sealers are available as handheld or benchtop models (Figure 17.7).

Donor tube stripper

A tube stripper (Figure 17.8a) is used to move fluid contained within the line connected to the blood collection pack back into that pack with minimal damage to the contents. Mixing of the contents of the pack should occur while the tube stripper is held in place, as fluid will be drawn back into the tubing once the stripper is released (Figure 17.8b).

Atraumatic clamps

Atraumatic clamps (Figure 17.9) are designed to allow temporary halting of fluid through the lines while not damaging the plastic tubing. A permanent seal, if required, can then be applied. Smaller plastic clips can also be used during centrifugation.

Component processing

The process of converting a WB collection into high-quality blood products is influenced by several factors including centrifuge settings and pre-storage conditions. Component processing is more commonly used for canine than feline products. While the principles for processing feline and canine blood donations are the same, the difference in scale restricts the range of feline blood products that can be readily manufactured. In addition, feline WB donations collected in a syringe and not transferred into a sterile plastic storage container cannot be processed as per canine collections in a commercial blood collection set. Thus the following paragraphs predominantly pertain to canine blood product manufacturing, while the special case of feline blood component processing is discussed separately (see the section on processing of feline blood products). Blood processing protocols are given in Box 2. Many issues can arise during blood processing; a guide to troubleshooting the most common issues is given in Table 17.1.

Table 17.1 Troubleshooting and tips on blood processing

Problem	Possible reason	Suggested solution
Uneven number of units to centrifuge		Use a water-filled blood collection bag as a balance Ensure bag is sealed Spare portions of plastic tubing or rubber bands act as small balances
The seal between the bags is broken before centrifugation	Accidental snapping of the seal	Place a plastic clamp on the tubing to temporarily seal the tubing (Figure 17.10)
Centrifuge shakes when starting	Unbalanced centrifuge	Ensure all centrifuge buckets and units are precisely weight balanced before centrifugation Major vibration: stop centrifuge and recheck balancing Minor vibration: can be normal during acceleration
Banging noises inside the centrifuge during centrifugation	Loose items in centrifuge	Immediately stop the centrifuge and check that all lines and bags are tucked securely away Check for any loose items within centrifuge
Cloudy or red plasma after centrifuging	Centrifuge bucket may have been knocked when removing from centrifuge, disturbing the layers Quick braking action will disturb the centrifuged layers RBCs might have hemolyzed due to extreme temperatures or a traumatic venipuncture Plasma might be lipemic	Check brake speed: turn off completely if necessary Re-centrifuge the units for up to one-third of the full centrifuge time If plasma is still red after a second centrifugation, then discard the whole pack as visible hemolysis means that the tolerated degree of hemolysis (0.8% of RBC) will likely be exceeded Lipemic plasma and RBCs can be used
Large pockets of RBCs at top of unit after centrifuging	Blood pack folded over before centrifugation, trapping RBCs	Gently resuspend the trapped RBCs while the unit is still in the centrifuge and re-centrifuge for up to one-third of the full centrifuge time Before starting centrifugation, check that the bags are sitting upright and not folded over
Indentations in donation bag after centrifugation	The plastic of the blood collection bag can be stretched during centrifugation by objects placed next to it	Wrap tubing inside satellite bags before placing in centrifuge bucket and ensure the flat surface of the satellite bag is next to the blood collection pack (Figure 17.13) Avoid placement of non-pliable objects next to blood collection pack, e.g. leukoreduction filters
Long bleeding time (>15 minutes)		The donation should not be used for production of platelets or plasma products for transfusion (Roback et al. 2011; European Committee on Blood Transfusion 2013) Clearly identify unit during processing and use RBCs only
No suitable centrifuge for centrifuging blood donations		Sit the unit upright in a refrigerator for 12–24 hours and then gently express the plasma off the settled RBCs If the plasma is removed, it should be replaced with additive solution Alternatively, use the product as a stored whole blood
Air bubble in plasma bag after expression of RBCs	Air is normally present within the blood collection bags, as they are not vacuum-packed The air is sterile if the blood collection technique has been performed correctly	Bubble can be left with the plasma as an indicator of accidental thawing Alternatively, transfer the bubble back onto the RBCs before physical separation and use to aid withdrawal of additive solution from transfer bag
Some additive solution still remaining in transfer bag	The seal between bags might not have been snapped	Check seals of both bags to ensure they are completely snapped Air in the RBCs can be expressed into the additive bag to aid extraction
One of the applied tubing seals is leaking	Faulty tubing seal application	Check equipment is operating correctly If the tubing seal is directly adjacent to the product, the closed system is potentially breached Use the product within 24 hours if during production or discard if noted during storage

RBC, red blood cell.

Box 17.2 Blood processing protocols

Preparation of the whole blood unit

1. The whole blood unit should be received for processing with the blood collection needle removed.
2. The tubing must be sealed at the completion of the donation to prevent air contamination of the unit. A line clamp can be used for this purpose, but must not be removed until a permanent seal is applied.
3. Blood in the collection line is stripped using a tube stripper and mixed with the anticoagulant in the bag immediately after the donation is complete. If this is not performed, the blood in the tubing will clot and might enter the product (Figure 17.10).

Figure 17.10 Blood clot in packed red blood cells (black arrow). This is due to inadequate line stripping and mixing immediately after blood collection. If unsure whether the line has been stripped, remove or seal the line at the top of the bag.

Whole blood (WB) processing

1. Turn centrifuge on and allow it to cool down to target temperature.
2. Weigh the WB collection, including satellite bags, and record weight.
3. Place the WB collection into your centrifuge bucket. Ensure the placement within the bucket meets recommendations by the centrifuge manufacturer, for example the WB should be placed towards the outside of a circular centrifuge bucket (Figure 17.11).
4. Fold the attached bags and lines securely into the bucket next to the unit (Figures 17.11 and 17.12). If using a set that contains a leukoreduction filter, ensure it is tucked away so that it will not cause damage to the unit while centrifuged.
5. Weigh the filled centrifuge buckets and select the heaviest bucket to balance all others against. The centrifuge buckets must be accurately balanced. Suitable balances include pieces of tubing, unused blood collection bags, or other soft plastics and rubbers.
6. Check that all the buckets swing freely in their axis (Figure 17.13).
7. Centrifuge the unit(s) following the appropriate protocol (Table 17.2).
8. During centrifugation, prepare all other equipment: plasma extractor, tube sealer, tube stripper, and scissors.
9. At completion of the centrifugation, carefully inspect the units to ensure the plasma is clear.
10. Carefully remove the bucket from the centrifuge, slowly remove the unit, place it in the prepared plasma press, and gently release the pressing plate.
11. Snap the seal at the top of the blood collection pack to allow plasma to run into the satellite bag (Figure 17.14).
12. For manual plasma presses, use thumb and index finger or an atraumatic tube clamp (Figure 17.9) to pinch the line shut while stopping the pressing plate from further plasma expression. Raising the plasma bag to the same level as the red blood cells can also assist to prevent further fluid transfer.
13. Seal the line between the red cells and plasma using the available method. Ensure the seal allows both the plasma and red cells to remain sterile once physically separated from each other.

Figure 17.11 Loading the blood collection pack into a centrifuge bucket (a) and centrifuge (b). Note that the heaviest items are placed towards the outside of the centrifuge.

Figure 17.12 Folding of satellite bag. Extra tubing is placed inside bag folds.

Figure 17.13 Checking free rotation capability of centrifuge bucket.

Table 17.2 Centrifuge RCF and times to obtain different blood products from whole blood (Roback et al. 2011)

Starting product	End product(s)	Settings	RCF and time
WB	PRBCs and plasma PRBCs and PRP BC, plasma, and PRBCs	Hard spin, 4°C (39.2°F) Soft spin, 20°C (68°F) Hard spin, 20°C (68°F)	5000 g, 7 minutes^a 2000 g, 3 minutes 5000 g, 7 minutes
PRP	Platelets and plasma	Hard spin, 20°C (68°F)	5000 g, 7 minutes
BC	Buffy coat platelets	Extra soft spin, 20°C (68°F)	700 g, 5 minutes
FFP (thawed at 1–6°C [33.8–42.8°F])	CRYO and CRYO-poor plasma	Hard spin, 4°C (39.2°F)	5000 g, 5 minutes

BC, buffy coat; CRYO, cryoprecipitate; FFP, fresh frozen plasma; g, unit of acceleration (equal to gravity); PRBCs, packed red blood cells; PRP, platelet rich plasma; RCF, relative centrifugal force; WB, whole blood.

^a Some authors recommend not exceeding centrifugation times beyond 5 minutes to reduce hemolysis (Sowemimo-Coker 2002).

Packed red blood cells

1. Process the blood as described for WB processing, using a hard spin.
2. Snap the seal between the additive solution (AS) and concentrated red cells.
3. Suspend the AS and allow the fluid to run onto the concentrated red blood cells.
4. Remove the AS bag if not required, using the method of tube sealing available and leaving a tubing segment long enough to later generate several sub-segments.
5. Gently mix to resuspend the red cells in the AS.
6. Use a tube stripper to strip the line content into the bag (Figure 17.8). Repeat three or four times to ensure a representative sample of the final red blood cell product remains in the line.
7. Subdivide the tubing into segments or “pig-tails” using the available method of tube sealing.
8. Remove a “pigtail” for measurement of final PCV and record value.
9. Weigh the unit if desired to establish approximated volume.
10. Label the bag according to local convention, then place in the refrigerator.

Platelets (from platelet-rich plasma [PRP])

1. Process the blood as described for WB processing.
2. Ensure the presence of a sterile satellite bag attached to the plasma bag and that this bag is suitable for platelet storage (e.g., PL2209).
3. Centrifuge the PRP using a hard spin.
4. Place the bag on the plasma extractor and remove most of the plasma, but leave approximately 50 mL of plasma with the platelet sediment.
5. Seal the tubing between the platelets and plasma using the available method.
6. Place the platelet concentrate on the bench top up to 60 minutes to allow platelets to disaggregate.
7. Gently resuspend the platelets in the plasma.
8. Label the bag according to local convention.
9. Place the platelets on a platelet rocker/agitator maintained at room temperature.

Plasma

1. Process the blood as for WB processing, using a hard spin.
2. Remove the plasma bag from the centrifuged red cells or platelets (PRP method).
3. Label the bag according to local convention and weigh if desired.
4. Place in a freezer at –18°C or colder, or a blast freezer. Lay the unit flat and in single layers to freeze as rapidly as possible.

Cryoprecipitate (CRYO)

1. Ensure that a sterile satellite bag is attached to the frozen FFP so that CRYO processing can occur.
2. Remove frozen FFP from the freezer and allow thawing at 1–6°C overnight.
3. Ensure the centrifuge is turned on and cold before processing.
4. Centrifuge the FFP using a hard spin at 4°C.
5. Place the bag on a plasma extractor and express the supernatant plasma into the attached satellite bag, allowing approximately 15 mL of plasma to remain above the CRYO sediment.
6. Separate and seal the bags using the available sealing method.
7. Label the bag according to local convention.
8. Immediately refreeze the CRYO and resulting CRYO-poor plasma at –18°C or colder.

Blood donation centrifugation

The optimal centrifuge speed for blood product processing depends on several factors. These include centrifuge specifications such as rotor size, but also the sedimentation characteristics of the desired blood components. These characteristics are also influenced by blood temperature, and protein and hemoglobin concentration (Roback et al. 2011). These are all factors that the operator has little control over, and therefore a first approximation to optimal centrifuge speed and time settings dictated by the basic principles outlined below must be followed by a second step during which the settings are adjusted to fulfil predefined quality control metrics (Table 17.2).

The main external factors determining the degree of sedimentation are the duration of centrifugation (time), the revolutions per minute (RPM), and the rotor size of the centrifuge (Roback et al. 2011). The operator has control over temperature, speed, brake time, and overall centrifugation time with a typical centrifuge. The total centrifugal force (TCF) is the overall force applied to the blood product during the acceleration and maintenance phase of the centrifugation process (Figure 17.15).

c17f015 — **Figure 17.15** Total centrifugal force (TCF) as area under the curve delineated by relative centrifugal force (RCF) integrated over the duration of centrifugation. After an acceleration phase (A to B), a phase of stable centrifugation (B to C) is followed by deceleration (C to D). TCF represents the overall effect of centrifugation. A reduction of RCF from 5000 g (dark grey area) to 2128 g (light grey area) requires a prolongation of centrifugation time if an identical TCF is to be achieved.

Centrifuge speed is expressed in either RPM or relative centrifugal force (RCF) and depends on the intended blood product. RCF is the force applied to the blood product under rotation at a specific point in time, and is directly influenced by the radius of the rotor in use, as well as the RPM. Deceleration is not included in the calculated time, and can be selected separately by adjusting the brake parameter on the centrifuge. Many centrifuges, particularly older styles, might not be designed to exceed 3000 RPM, and only offer settings for RPM and not RCF. Recommendations for component centrifugation are provided in RCF, such that formulae are required for conversion into RPM settings (Box 3). It is important to remember this can serve as a guideline only. Minor adjustments in time and/or speed for a specific centrifuge will be required and variations in acceleration time need to be factored in. A trial period will therefore be necessary before optimum settings can be established for a particular centrifuge. A service engineer or representative of the centrifuge manufacturer should be able to assist further if required.

Tags: Manual of Veterinary Transfusion Medicine and Blood Banking

Sep 27, 2017 | Posted by admin in GENERAL | Comments Off

Veterian Key

Fastest Veterinary Medicine Insight Engine