Improving Blood Banking Protocols Using the RC3BP Plus

Application Note: AN-LECF- Improving Blood Banking Protocols BBPROTOCOL-0508 using the Thermo Scientific RC3BP Plus™

Hema Downey, Newtown, CT 06470 Nadia Boujtita, PhD, Laboratory Equipment Division, Thermo Fisher Scientific

Introduction Second spin: The first manufacture optimization of the centrifugation KEY WORDS Blood banks optimize their of the platelet-rich plasma conditions allow leukocyte-depleted protocols to yield the highest is followed by the second spin platelets to be produced by the • Blood Banking quality products in the shortest at 5,000 x g for 5 min obtaining buffy coat method. • Platelet Concentrate possible time. Optimized protocols the platelet concentrate. Preparation may vary from blood bank to During the second spin, platelets Why Use the Thermo Scientific blood bank depending on which are pelleted from the PRP to yield Sorvall RC3BP Plus • Low Speed Centrifuge blood products are most desired. platelet concentrate and platelet The Thermo Scientific Sorvall This document is a simple, step- poor plasma. In choosing the length RC3BP Plus centrifuge improves by-step guide to troubleshooting and speed of the second spin, productivity for low-speed blood low product yields. platelets should be pelleted tightly, bank applications. Also, the Whole blood collection volumes but not to the point of aggregation. RC3BP Plus centrifuge offers (405 – 495 mL for 450 mL bags; If this happens, the platelet yield a proven set of swinging-bucket 450 – 550 mL for 500 mL bags) will appear low because the cells rotors that meet the specific needs vary, affecting the volume of plasma, stick together and the unit can not of the blood banking market. A red cells and platelets. Donor-to- be used. Also, platelet contaminants summary of centrifuge conditions donor variability leads to platelet in the plasma can decrease the using Thermo Scientific instruments concentrate variations, e.g. low antihemophilic factor (AHF) yield; for the preparation of platelet platelet donors give lower yields therefore the plasma should be concentrate from platelet-rich of platelet concentrate. So how does as cell-free as possible. plasma is listed in Table 1. one start to establish protocols with all these known variables? Preparation of Platelet Concentrate from Buffy Coat Preparation of Platelet Concentrate Platelets may also be prepared from Platelet-Rich Plasma from buffy coat, a method used Following the calibration of mainly in Europe. centrifuges for platelet manufacture First spin: After centrifugation in the AABB Technical Manual; for 7 min at 5000 x g, whole the AABB suggests the following blood is separated into red blood centrifuge conditions: cells (RCBs), buffy coat containing Thermo Scientific H-6000A swinging- First spin: Separates whole blood the platelets and platelet-poor bucket rotor unit into platelet rich plasma (PRP), plasma (PPP). The buffy coat is buffy coat (WBC’s) and red blood separated and further processed cells (RBCs) by centrifugation to obtain a platelet concentrate. at 2000 x g for 3 min. Either single buffy coat or 4 to The first centrifugation step 6 pooled (blood group compatible) for platelet production is critical. buffy coats are diluted with plasma If the first spin is too long and or an appropriate nutrient solution. hard, platelets will be lost to the Second spin: The buffy coat or buffy coat and the red blood cells buffy coat pool is then centrifuged resulting in low platelet yields. for 3 min at 2000 x g to concentrate Conversely, too short a run does the platelets. not provide enough time to separate Leukocyte-depleted platelets an adequate plasma volume from can be prepared by filtration, the whole blood. This results pre-storage leukocyte depletion in a reduced volume of plasma is recommended (preferably within Thermo Scientific Sorvall RC3BP Plus products and bloody platelets. 6 hours after recovery). Careful centrifuge Rotor RCF (x g) Speed (rpm) Time (min) Effect (ACE™) function, a standard 1st Spin: feature of RC3BP Plus. This H-6000A 2,000 2,600 3 function assures you of repro- HBB-6 2,000 2,650 3 ducible blood product yields, HLR-6 2,000 2,635 3 regardless of rotor loading. The ACE function calculates the area 2nd Spin: under the Speed/Time curve as H-6000A 5,000 4,150 5 shown in Figure 1. This is the HBB-6 5,000 4,200 5 Accumulated Centrifugal Effect HLR-6 5,000 4,165 5 experienced during a centrifugation run, by particles in a solution, Table 1. Centrifuge conditions for the first and the second spins for platelets production when such as platelets in whole blood. using Thermo Scientific Sorvall RC3BP Plus with either H-6000A, HBB-6 or HLR-6 rotors. Processing temperature range: 22 ± 2°C. Using the ACE Mode of Sorvall RC3BP Plus ACE automatically compensates Multiple Thermo Scientific How to Maximize Blood Product Yields for variations in acceleration due Rotors for Blood Banking In slow start/stop mode, you can to full or partial rotor loading by The H-6000A Swinging-Bucket select from 11 acceleration and adjusting the centrifuge run time. Rotor offers flexibility for multiple deceleration profiles to customize An ACE value and speed is protocols. It accommodates up results for maximum yields with set at the start of a run, allowing to 6 single, double, triple or quad minimal re-suspension. the centrifuge to control the blood bags. There is a wide range Accumulated Centrifugal Effect of adapters for blood collection How to Improve Blood Product up to deceleration (see Figure 2, tubes, conical tubes and bottles. Reproducibility 2,800 rpm and an ACE value The HBB-6 Swinging-Bucket As the first centrifugation step of 1.60 x 107). As observed in the Rotor can be used to process for platelet production is critical, graph below, times were adjusted a wide range of blood bag types. to ensure run-to-run reproducibility to achieve the same overall g-force It accommodates up to 6 whole use the Accumulated Centrifugal regardless of the rotor load. blood bags, in configurations from singles to quintuples. There is a large choice of adapters for up to 12 platelet-rich plasma and buffy-coat bags. The HLR-6 Swinging-Bucket Rotor incorporates a separate filter holder to preserve blood bag integrity. It was optimized for soft- spin production of leuko-reduced packed red cells and platelet concentrates. The HLR-6 accommodates up to 6 triple, quad or quint blood bags with in-line filters. Figure 1. Speed/Time Curve illustrates a typical first centrifugation step (2,800 rpm) with a two-bag rotor load versus six. Notice the two-bag load attains set speed faster than the six-bag load. In a time mode, since both loads will time out at the set time of 3:30 minutes, How to Adjust Your Protocol they experience different gravitational forces (g-force). In order to obtain the same overall Make adjustments in the speed g-force for both loads, change the time for 2 bags to 3:00 minutes or use the ACE function. by 200 rpm increments or time by 30 seconds. Adjust the protocol until the desired yield of products is obtained. • If the platelet yield is low or plasma volume high, decrease the time or lower the speed for the first spin. • If the plasma yield is low, increase the time or speed for the first spin. More details are described on the Table 2 “Troubleshooting Guide to Improve Blood Product Yields”. Figure 2. Speed/Time Curve with ACE function Problem/ 1st Spin 1st Spin 2nd Spin 2nd Spin In addition to these offices, Observation Finding Action Finding Action Thermo Fisher Scientific maintains a network of Platelet pellet appears OK Keep speed and time as is OK Keep speed and time as is representative organizations firm, well packed throughout the world. Platelet concentrate OK Keep speed and time as is Too hard Decrease time or speed has aggregates present Platelet pellet appears OK Keep speed and time as is Too soft Increase time or speed soft, loosely packed Plasma and red cell OK Keep speed and time as is OK Keep speed and time as is volume acceptable Plasma volume high, Too hard Decrease time or speed OK Keep speed and time as is red cell volume low Plasma volume low Too soft Increase time or speed OK Keep speed and time as is Platelet yield and plasma OK Keep speed and time as is OK Keep speed and time as is volume acceptable North America: Platelet yield is low Too hard Decrease time or speed OK Keep speed and time as is USA / Canada +1 866 984 3766 and pellet appears firm Europe: Platelet yield is low Too hard Decrease time or speed Too soft Increase time or speed Austria and pellet appears soft +43 1 801 40 0 Belgium Platelet yield acceptable, Too soft Increase time or speed OK Keep speed and time as is +32 2 482 30 30 plasma volume low France No distinct red cell Too hard Decrease slow stop rate OK Keep slow stop rate same +33 2 2803 2000 China and plasma line. +86 21 6865 4588 or ‘Bloody interface’ +86 10 8419 3588 Germany national toll free 08001-536 376 Table 2. Troubleshooting Guide to Improve Blood Product Yields Germany international +49 6184 90 6940 Italy +39 02 02 95059 434-254 Conclusion References Netherlands The Thermo Scientific Sorvall 1. AABB Technical Manual. +31 76 571 4440 Nordic countries RC3BP Plus centrifuge provides 12th Edition 1996. +358 9 329 100 a complete solution for low speed 2. Establishing Blood Banking Russia / CIS applications in blood bank Protocols on the RC3BP and +7 (812) 703 42 15 Spain/Portugal laboratories, delivering reliable RC12BP. Application Brief # +34 93 223 09 18 and reproducible results. S00052. Switzerland +41 44 454 12 12 3. Blood Component UK/Ireland Manufacturing: In-Process +44 870 609 9203 Control Using ACE. Asia: China Application Brief # S00297. +86 21 6865 4588 or +86 10 8419 3588 India +91 22 6716 2200 Japan +81 45 453 9220 Other Asian countries +852 2885 4613 Countries not listed +49 6184 90 6940 or +33 2 2803 2000

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