BD Leucocount™ and BD™ Plasma Count Reliable Enumeration of Residual Cells in Blood Products

• Improve the efficiency of your Quality Control assays. • Discover high-performance flow cytometric methods for enumerating residual cells in your blood products.

Residual cell contamination in blood-derived products is associated with an increased incidence of febrile transfusion reactions, cytomegalovirus (CMV) transmission and alloimmunization to HLA antigens.1-3 Leucodepleted blood products minimize transfusion-associated complications and are the standard for supporting patients in need of multiple transfusions.4-6 Advancements in leucodepletion technology have led to decreased residual cell levels, frequently resulting in concentrations lower than the sensitivity range of traditionally used counting methods. As European guidelines for the leucoreduction of blood components are systematically being implemented, requirements for setting up fast and reliable BD™ Plasma Count process control programs to monitor residual cell levels in blood- derived products are becoming increasingly important. Simultaneous Enumeration of Residual White Blood Cells, Red Blood Cells and Platelets Flow cytometry provides a rapid, sensitive, quantitative and reproducible application to identify and enumerate residual 50 tests Cat. Nr. 338331 cell populations.7-12 BD Leucocount™ and BD™ Plasma Count are flow cytometric assays, designed for counting residual cell populations in blood component samples. The BD Leucocount and BD Plasma Count kits are available as CE-marked products and are sufficient for performing 50 tests.

BD Leucocount™

Residual White Blood Cell Enumeration Kit

50 tests Cat. Nr. 340523 A system you can Count on. The NEW BD Plasma Count assay provides an easy-to-use method for the simultaneous quantitation of residual white BD Leucocount and BD Plasma Count offer you efficient blood cells (rWBCs), red blood cells (rRBCs) and platelets alternatives to tedious and operator-dependent manual methods (rPLTs) in fresh plasma. All three residual cell populations for cell enumeration. Both assays incorporate our BD Trucount tubes to determine absolute cell counts, thereby eliminating the are identified and enumerated in one single assay, through need for separate counting standards as well as the inaccuracies multi-parameter staining and analysis. By using the same associated with the pipetting of liquid bead suspensions. reagents and blood component sample for all three results, Both assay procedures can be readily implemented onto your the BD Plasma Count assay protocol reduces the risk of existing flow cytometer. Together with your instrument, errors in sample preparation and counting operations, BD Leucocount and BD Plasma Count reagents provide you with allows for the efficient use of reagent, and limits the volume a high-performance system, to manage the Quality Control of blood product sample required for testing. process of your blood-derived products.

The BD Leucocount assay is designed for counting residual white blood cells (rWBCs) in leucoreduced blood products for use in transfusion, such as platelet and red Reliable. Compliant. Fast. cell packs. The BD Leucocount kit has been engineered to The BD Leucocount and BD Plasma Count assays are accurate, provide an effective and sensitive method for enumerating stable and robust. They provide excellent linearity, precision and rWBCs while eliminating limitations associated with other accuracy, at clinically relevant decision thresholds in compliance techniques.13,14 with European recommendations for the preparation, use and quality assurance of blood components.15 BD Leucocount and BD Plasma Count give you the highest level Straightforward procedures. of confidence in your process control. There is no further need for spending long hours performing The BD Leucocount and BD Plasma Count assays save you time manual cell counts. Automation and high-throughput are and labor, with their fast, simple procedures: achievable with both BD Leucocount and BD Plasma Count BD Leucocount methods, through combining them with the BD FACSCalibur™ flow cytometer and the BD FACS™ Loader option. • Add 100 µl of sample to a BD Trucount™ Tube. The ease-of-use of the BD Leucocount and BD Plasma Count • Add 400 µl of BD Leucocount reagent. assay systems ensures that quality data are obtained, even in the • Mix, incubate for 5 minutes, then acquire and analyze with busiest blood centers. BD CellQuest™ Pro software.

BD Plasma Count • Add 25-50 µl* of sample to a BD Trucount Tube. • Add 100 µl of Reagent Mix A. • Add 20 µl of Reagent Mix B. • Mix, then incubate for 15 minutes in the dark. • Add 1 ml of BD CellWASH™ solution, mix, and analyze with BD CellQuest Pro software.

*depending on range of rWBCs in sample

The BD Leucocount and BD Plasma Count kits contain reagents sufficient for performing 50 tests.

BD FACSCalibur flow cytometer Optimized reagents.

The BD Plasma Count Kit is optimized for the simultaneous The BD Leucocount Kit uses a single reagent for both platelet detection and quantitation of three residual cell populations and red cell products. – rWBCs, rRBCs and rPLTs – in fresh human plasma, in a The BD Leucocount reagent contains: convenient single tube assay. • A Nucleic Acid Dye BD Plasma Count reagents include: Propidium Iodide (PI), for staining all nucleated cells. This DNA/ • Reagent Mix A RNA-specific dye is excited at 488 nm and emits in the FL2 and Thiazole Orange, for staining all nucleated cells. Thiazole FL3 channels. Brightly stained leucocytes are detected in FL2 Orange is a membrane-permeable nucleic acid dye that and are easily distinguishable from any non-nucleated particles, binds DNA and RNA. Excited at 488 nm, it emits in all three such as erythrocytes and platelets. fluorescence channels on the BD FACSCalibur flow cytometer • RNase i.e. FL1, FL2 and FL3. The brightly stained leucocytes, easily for enzymatic digestion of any RNA present in the sample that distinguishable from any non-nucleated particles, appear along could otherwise be stained by PI and lead to falsely elevated a diagonal axis in all double-fluorescence correlated plots counts (i.e. FL1 vs. FL2, FL1 vs. FL3 and FL2 vs. FL3). They are well separated from the other cell populations and from the • A Detergent BD Trucount bead population. for permeabilizing the cell membrane to permit entry of PI

• Reagent Mix B • Buffers FITC-labeled anti-CD235a (Glycophorin A) antibody clone GAR- for providing a stable solution for stained samples, and for 2 (HIR-2), for staining rRBCs, and PerCP-Cy5.5-labeled CD41a optimizing fluorescence and scatter properties antibody clone HIP8, for staining rPLTs. FITC-stained rRBCs are • BD Trucount tubes detected in the FL1 channel, and PerCP-Cy5.5-stained rPLTs in for absolute counting of residual cells the FL3 channel on the BD FACSCalibur flow cytometer.

• BD Trucount tubes for absolute counting of residual cells. BD Trucount tubes contain a lyophilized pellet of fluorescent beads that serve as an internal standard for calculating absolute counts.

Figure 1. Representative plot summarizing the gating strategy used for BD Plasma Count data analysis. Region 1 contains the BD Trucount bead population. Regions 2, 3 and 4 contain the rWBC, rRBC and rPLT populations, respectively. Figure 2. Accuracy of the BD Plasma Count assay versus the Fuchs-Rosenthal manual counting method. In addition, associated process controls are available and offered References in different formats to best suit your laboratory’s Quality Control 1. Orlin J.B. and Ellis M.H. (1997). Transfusion-associated graft-versus-host program: disease. Curr Opin Hematol. 4:442. 2. Raife T.J. (1997). Adverse effects of transfusions caused by leukocytes. J • BD Leucocount RBC Control (25 tests – Cat. Nr. 341001) Intraven Nurs. 20:238. and BD Leucocount PLT Control (25 tests – Cat. Nr. 341002) 3. Wagner S. (1997). Transfusion-related bacterial sepsis. Curr Opin are composed of mammalian erythrocytes and platelets in a Hematol. 4:464. plasma-like fluid with preservatives, respectively, and spiked 4. de Graan-Hentzen Y.C., Gratama J.W., Mudde G.C. et al. (1989). with a known amount of white blood cells. Prevention of primary cytomegalovirus infection in patients with hematologic malignancies by intensive white cell depletion of blood • BD Leucocount RBC and PLT controls are also available products. Transfusion 29:757. together in a combination kit: BD Leucocount Combo 5. Sniecinski I., O’Donnell M.R., Nowicki B. et al. (1988). Prevention of Control Kit (25 tests each – Cat. Nr. 341003). refractoriness and HLA-alloimmunization using filtered blood products. Blood 71:1402.

These quality controls are in vitro diagnostic reagents, made of 6. Wenz B., Gurtlinger K.F., O’Toole A.M. et al. (1980). Preparation of stable material. They provide you with a means of monitoring granulocyte-poor red blood cells by microaggregate filtration: a simpli- the laboratory procedure with BD Leucocount for a high degree fied method to minimize febrile transfusion reactions. Vox Sang. 39:282. of confidence. 7. Frey B., Furrer M., Wettstein M. et al. (2001). Quantification of residual red blood cells in platelet concentrates and fresh frozen plasma by flow cytometry. Infusion Therapy and Transfusion Medicine 28 Suppl 1:53

8. Jilma-Stohlawetz P., Marsik C., Horvath M. et al. (2001). A new flow cytometric method for simultaneous measurement of residual platelets and RBCs in plasma: validation and application for QC. Transfusion 41:87.

9. Krailadsiri P. and Seghatchian J. (2001). Residual red cell and platelet content in WBC-reduced plasma measured by a novel flow cytometric method. Transfus Apher Sci. 24:279.

10. Lambrecht B., Spengler H.P., Bauerfeind U. et al. (2001). Simultaneous quantitation of contaminating leucocytes, erythrocytes, and platelets in fresh frozen plasma in a single tube assay by flow cytometry. Infusion Therapy and Transfusion Medicine 28 Suppl 1:52.

11. Pichler J., Printz D., Scharner D. et al. (2002). Improved flow cytometric method to enumerate residual cells: minimal linear detection limits for platelets, erythrocytes, and leukocytes. Cytometry 50:231.

12. Vowells S., Cadden M., Wagner C. et al. (1998). Detection and Enumeration of Residual White Blood Cells in Leucoreduced Red Blood Cell and Platelet Products Using the Leucocount Kit. BD Biosciences White Paper.

13. Rebulla P., Porretti L., Bertolini F. et al. (1993). White cell-reduced red cells prepared by filtration: a critical evaluation of current filters and methods for counting residual white cells. Transfusion 33:128.

14. Vachula M., Simpson S.J., Martinson J.A. et al. (1993). A flow cytometric method for counting very low levels of white cells in blood and blood components. Transfusion 33:262.

15. Council of Europe (2006). Guide to the preparation, use and quality assurance of blood components - 12th edition.

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