The leucoreduced PLT and RBC specimens were enrolled per the pre‐defined inclusion and exclusion criteria and were prepared using the BD Leucocount™ kit. Samples were measured on the BD FACSVia and BD FACSCalibur instruments side‐by‐side at four study centers to determine the WBC absolute count on each system. Table 1 lists the number of specimens enrolled at each study site.
Leucocount kit
Manufactured by BD
Sourced in United States
The BD Leucocount™ kit is a laboratory equipment product designed for the enumeration of white blood cells (leukocytes) in biological samples. It provides a standardized and reliable method for quantifying the number of leukocytes present in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (2 mentions)
Trucount tube, by BD (2 mentions)
Facsvia, by BD (1 mentions)
Macsquant 10, by Miltenyi Biotec (1 mentions)
Macsquantify software, by Miltenyi Biotec (1 mentions)
Facsvia system, by BD (1 mentions)
Cobas 8000 ise module, by Roche (1 mentions)
Facscanto 2 cytometer, by BD (1 mentions)
Plasma low hb photometer, by HemoCue (1 mentions)
6 protocols using leucocount kit
1
Evaluation of WBC Counting Systems
2
Leukocyte Quantification in Cell Samples
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Donor samples, PLT and RBC controls were prepared using BD Leucocount Kit (BD Bioscience, San Jose, CA, USA), according to the manufacturer´s instructions. Briefly, 100 μL of well-mixed cell sample was dispensed into a Trucount tube (BD Bioscience, San Jose, CA, USA), which contained a predefined number of counting beads. Cells were labeled by adding 400 μL of Leucocount reagent to each tube. The reagent contained a nucleic acid dye propidium iodide (PI) and RNAse, and therefore only cellular DNA was stained. The tubes were gently mixed and incubated for 5 min in the dark at room temperature. All PLT and RBC control cells were treated in the same way as specimen samples.
After staining, samples were analyzed with the FACSCalibur (BD Biosciences, San Jose, CA, USA), using the CellQuest Pro software and a BD template for residual leukocytes enumeration, and the MACSQuant 10 (Miltenyi Biotec, Bergisch Gladbach, Germany), using the MACSQuantify software (Miltenyi Biotec, Bergisch Gladbach, Germany) and a modified in-house template (time/leukocytes and time/beads, seeS1 Fig ). Ten thousand events of the bead population were recorded at low flow speed and analyzed.
The absolute leukocyte count was determined using the following formula:
After staining, samples were analyzed with the FACSCalibur (BD Biosciences, San Jose, CA, USA), using the CellQuest Pro software and a BD template for residual leukocytes enumeration, and the MACSQuant 10 (Miltenyi Biotec, Bergisch Gladbach, Germany), using the MACSQuantify software (Miltenyi Biotec, Bergisch Gladbach, Germany) and a modified in-house template (time/leukocytes and time/beads, see
The absolute leukocyte count was determined using the following formula:
3
Inter-laboratory Reproducibility of PLT and RBC Control Cells
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Custom made PLT and RBC control cells (R&D Systems, Minneapolis, MN) were used as a stable cellular material to evaluate inter‐laboratory reproducibility for the BD FACSVia System. Two concentration levels of PLT control cells, 7.6 ± 2.7 and 17 ± 4.3 cells/µl, and two concentration levels of RBC control cells, 7.2 ± 2.5 and 19 ± 4.8 cells/µl, were used. Controls were shipped from BD Life Sciences, San Jose to the study sites. Each of the three study sites stained each concentration level sample of PLTs and RBCs in duplicate in BD Trucount tubes, using the BD Leucocount kit, and the resulting stained samples were run on a BD FACSVia System.
4
Preparation and Validation of Sheep Blood Components
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The preparation and validation of blood components has previously been described in detail [19 (link)]. Briefly, two units of whole blood (WB; 1 unit = 450 ml ± 10% v/v) were collected from each donor sheep into blood packs (Fresenius Hemocare NPBI), containing CPD.A1 anticoagulant. Components were separated by centrifugation and extraction using a Compomat G4 (Fresenius Hemocare) optimised for the separation of sheep blood components, to yield platelet-rich plasma, red cells and buffy coat. Platelet-rich plasma was further centrifuged to produce platelet-poor plasma and platelet concentrate. Leucodepletion of components was performed using in-line leucoreduction filters (Fresenius/NPBI).
Prior to transfusion, 10–20 ml of each component was sampled for analysis and archive storage/bioassay. For each component, the volume was estimated based on weight (adjusted for specific gravity). Numbers of leucocytes in non-leucodepleted and leucodepleted components were measured by flow cytometry, using a Leucocount kit (BD Biosciences). Numbers of platelets in platelet concentrates and plasma fractions were counted manually using a haemocytometer. In whole blood, red cells and buffy coat, the haematocrit was measured, using a haematocrit centrifuge and reader (Hawksley), to determine the distribution of plasma in different components.
Prior to transfusion, 10–20 ml of each component was sampled for analysis and archive storage/bioassay. For each component, the volume was estimated based on weight (adjusted for specific gravity). Numbers of leucocytes in non-leucodepleted and leucodepleted components were measured by flow cytometry, using a Leucocount kit (BD Biosciences). Numbers of platelets in platelet concentrates and plasma fractions were counted manually using a haemocytometer. In whole blood, red cells and buffy coat, the haematocrit was measured, using a haematocrit centrifuge and reader (Hawksley), to determine the distribution of plasma in different components.
5
Comprehensive Blood Analysis Protocol
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Hemoglobin, hematocrit, platelet count, and WBC were analyzed on a Cell-Dyn Sapphire analyzer (Abbot Diagnostics, Abbot Park, IL). The Leucocount kit from BD (BD Biosciences, San Jose, CA) was used for residual WBC analysis, with a BD FACSCanto II cytometer. Plasma hemoglobin was analyzed with the HemoCue Plasma/Low Hb photometer (HemoCue AB, Angelholm, Sweden) to calculate hemolysis.
Lactate and blood gas analysis was performed on whole blood samples with an ABL 800 FLEX (Radiometer Medical ApS, Brøndshøj, Denmark).
Glucose, potassium, and Factor VIII were analyzed with the Cobas 8000 ISE module (Roche Diagnostics GmbH, Rotkreuz, Switzerland). FVIII and fibrinogen levels were analyzed with the STA-R Evolution/STA-R Max platform (Stago S.A.S., Asnieres-sur-Seine, Paris, France).
All plasma samples were prepared by centrifugation at 1850 G for 10 min in RT.
Lactate and blood gas analysis was performed on whole blood samples with an ABL 800 FLEX (Radiometer Medical ApS, Brøndshøj, Denmark).
Glucose, potassium, and Factor VIII were analyzed with the Cobas 8000 ISE module (Roche Diagnostics GmbH, Rotkreuz, Switzerland). FVIII and fibrinogen levels were analyzed with the STA-R Evolution/STA-R Max platform (Stago S.A.S., Asnieres-sur-Seine, Paris, France).
All plasma samples were prepared by centrifugation at 1850 G for 10 min in RT.
6
Efficient Platelet-Rich Plasma Isolation
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EDTA-anticoagulated venous blood was collected and platelet-rich plasma (PRP) was recovered by centrifugation at 150g for 15 minutes. The upper third of the PRP was recovered and an additional centrifugation procedure removed the remaining red (RBC) and white blood cells (WBC), as described elsewhere (Burkhart et al. 2012; Raghavachari et al. 2007; Zufferey et al. 2014) . The amount of residual WBC and RBC was evaluated by microscopic inspection and FACS analysis using the Leucocount kit (BD Biosciences, Franklin Lakes, USA, reference 340523) (Zufferey et al. 2014) . This method yielded 0.8 WBC per 10 6 platelets) and 1 RBC per 540,000 platelets, in line with previous reports (Burkhart et al. 2012; Kaiser et al. 2009; O'Neill et al. 2002 ). An additional evaluation of WBC contamination was performed using gene expression levels as described in the Online Data Supplement (Supplementary Fig. 1) and with western blot analysis showing no CD11 (a marker of WBC) in our samples (data not shown).
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