Application Note Efficient buffy coat extraction Reliable extraction of buffy coat from human blood on a Freedom EVO® platform Introduction The recent advances in personalized diagnostics continue to A valuable body fluid for diagnostic purposes is the buffy coat, inspire a wealth of personalized treatments, resulting in a layer of leukocytes and platelets that forms between the state-of-the-art personalized heathcare. The Integrated erythrocyte layer and the plasma when unclotted blood is Biobank of Luxembourg (IBBL), a newly founded independent, centrifuged or allowed to stand. Typically, the isolation of the not-for-profit biobank, designed for a new era of research and buffy coat from whole blood has been a tedious and lengthy the next generation of healthcare, is at the forefront of these manual process. Researchers at the Integrated Biobank of medical advances. Research into personalized medicine Luxembourg have tested and evaluated the feasibility and requires high quality samples (including tissue and body efficiency of extracting the buffy coat using an automated fluids, such as blood, blood fractions and saliva) and clinical process on Tecan’s Freedom EVO platform. data from large numbers of patients, collected by biobanks, to translate today’s discoveries into future personalized medical The automation of buffy coat extraction is challenging. In care innovations. As a world class EU biobank, the IBBL addition to the blood volumes in the individual blood collection provides a wide variety of the highest quality samples, tubes varying, important inter-individual variations in the alongside cutting-edge technology, to accelerate clinical quantity, viscosity and texture of the buffy coat layer itself, for research and improve public health. example due to leukocyte increase upon infection, can be observed. Tecan’s Freedom EVO platform, equipped with a Tube Inspection Unit (TIU), has been optimized for automated buffy coat extraction, giving superior results compared to the manual process. 1 Application Note Materials and Methods Plasma Instrument Tecan Freedom EVO 200 liquid handling workstation with an eight-channel Liquid Handling (LiHa) Arm, equipped with: Upper fraction limit Module Module description/benefit Buffy Coat Pipetted fraction Pick and Place Arm Moves tubes Separation layer (measured) (PnP) Lower fraction limit Tube Inspection Unit Identifies separation level (TIU) between serum and blood cells Red Blood Cells Robotic Manipulator Moves plates (RoMa) Arm Figure 2 Schematic diagram of fractionated blood. Xtr-96 flatbed scanner Tracks destination tubes (FluidX) Manual workflow Xsd-96Pro Decaps and recaps destination Manual buffy coat extraction was performed by slowly (FluidX) tubes aspirating 940 µl of buffy coat with a pipette in a circular Xsd-48Pro Decaps and recaps destination movement, trying not to disturb the red blood cell layer. FluidX) tubes BDK module HEPA filtered air on worktable Automated workflow The worktable layout is shown in Figure 1. The centrifuged decapped blood collection tubes were placed onto the Freedom EVO 200 platform and transferred to the Tube Inspection Unit, which identified the position of the buffy coat layer using a laser beam. The Liquid Handling Arm aspirated 940 µl of the buffy coat in a spiral movement (Figure 3), and transferred this fraction to the destination tube. The configuration and scripting of the Freedom EVO workstation were optimized to maximize the yield and quality of the buffy coat. Automated extraction of buffy coat from 24 samples Figure 1 Freedom EVO worktable, optimized for buffy coat (BC) extraction, with Tube Inspection Unit. takes just 16 minutes (Table 1). Sample preparation Automated process sample throughput K2-EDTA (BD) 10 ml blood collection tubes were centrifuged Sample numbers 1-24 samples at 2,000 g for 10 minutes at room temperature to separate the Batch time +/-16 mins for 24 samples Table 1 Overview of sample throughput for buffy coat extraction. blood into plasma, red blood cells and the intermediate thin, For larger batch sizes the time used increases proportionally. white colored layer, the buffy coat (Figure 2). 2 Application Note DNA yield After carefully optimizing the scripting for automated buffy coat extraction on the Freedom EVO, the efficiency of the extraction was confirmed by purifying DNA from the buffy coat. Buffy coat from 8 ml blood samples from approximately 100 donors was extracted using either the automated or the manual method and the DNA was purified. The automated extraction process demonstrated higher DNA yields compared to the manual extraction method (Table 3). 8 ml blood DNA yield (µg) Median 25 % 75 % n Figure 3 Automated pipetting of the buffy coat is performed in a spiral Manual BC 145.2 111.5 184.2 53 manner, with a defined movement from top to bottom. extraction Results Automated BC 199.1 181.6 239.3 49 extraction Table 3 Automated extraction of buffy coat gave a higher yield of DNA Verification of white blood cell populations compared to the manual process. To ensure that the buffy coat extracted using the automated process contains normal levels of white blood cell (WBC) Validation populations, flow cytometry analysis was performed on buffy To check the efficiency of buffy coat extraction, blood coat extracted from three different healthy donors (Table 2). collection tubes were inspected by eye after the withdrawal of Normal subpopulation percentages of WBCs were detected. equivalent volumes of buffy coat by the manual and automated extraction processes. The buffy coat withdrawal Population Automated Automated Automated appears more complete with the automated process percentages BC#1 BC#2 BC#3 (Figure 4). Lymphocytes 33.9 39.4 37.1 (% all cells) Monocytes 6.5 6.7 9.8 (% all cells) Granulocytes 59.6 53.9 53.1 (% all cells) CD3 T cells (% 66.7 72.9 67.1 lymphocytes) CD19 B cells 11.1 10.9 14.3 Manual BC extraction Automated BC extraction (% lymphocytes) Figure 4 Blood collection tubes after withdrawal of equivalent Table 2 White blood cells in buffy coats extracted using the automated volume of buffy coat by manual process (3 tubes on the left) process contain normal subpopulation percentages. Flow cytometry or automated process (3 tubes on the right). experiments were performed on a BD Influx™ cytometer using standard antibodies. 3 Application Note To check the efficiency of buffy coat extraction by a more process the numbers of white blood cells and platelets were quantitative approach, the blood cells in the buffy coat were higher, and red blood cell contamination and hemoglobin counted and compared to the cell numbers in whole blood levels were lower compared to manually extracted buffy coat. (Table 5). In the buffy coats extracted by the automated Whole blood Automated BC extraction Manual BC extraction Parameter measured Total cell number Total cell number Recovery (%) Total cell number Recovery (%) or quantity or quantity versus whole blood or quantity versus whole blood Mean Mean Mean WBC (106) 47.2 45.0 95 26.6 56 RBC (106) 36107 7405 21 9020 25 HGB (g) 1.088 0.225 21 0.271 25 PLT (106) 1931 172 69 676 35 Table 5 Blood cell counts for whole blood, and automated and manual extraction of buffy coat. WBC (white blood cells), RBC (red blood cells), HGB (hemoglobin), PLT (platelets). Conclusion Automation of buffy coat extraction on the Freedom EVO Talk to your local Tecan consultant to find out more about allows fast, convenient and reproducible extraction. customizing the Freedom EVO workstation to your • The DNA yield from the buffy coat extracted with the laboratories needs. automated method is higher compared to the manual method. For more information on the use of automation in biobanking, • Automation of buffy coat extraction permits the isolation visit www.tecan.com\biobanking of all leukocyte subpopulations. • Compared to manual buffy coat extraction, automation guarantees high sample throughput and reproducibility, eliminates operator variability, and automates sample tracking for secure identification. Acknowledgements For the highest flexibility and to meet changing laboratory Data was kindly provided by Conny Mathay, Wim Ammerlaan needs, the Tecan Freedom EVO workstation can be equipped and Fay Betsou, Integrated Biobank of Luxembourg, 6 rue with a number of extension modules. Nicolas Ernest Barblé, L-1210 Luxembourg, Luxembourg. 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