D t ti g Mi Detecting Microparticles p ti l in i Blood Bl d Components C p t – A Potential P t ti l Solution S l ti 1 2 , David 1,2 A d Audrey Labrie L b i 1,J Jennifer if Leung L D id C Crettaz tt 3, Michel Mi h l Prudent P d t3, Niels Ni l Lion Li 3 and d 12 Eli b h M Elisabeth Maurer-Spurej Maurer Spurej S j1,2 1LightIntegra Li htI t T Technology, h l Vancouver, V Canada, C d 2The Th U University i it off B British iti h C Columbia l bi Centre C t for f Blood Bl d Research, R h Vancouver, V Canada C d and d 3Service S i Régional Ré i l Vaudois V d i de d T Transfusion f i S Sanguine, i L Lausanne, Switzerland S it l d Background ‒ Transfusion T f i off blood bl d components t with ith high hi h microparticles i ti l (MP) loads l d (cell-derived (cell ( ll derived d i d or lipid) li id) can cause severe complications li ti in i patients, ti t however h measuring i the th MP load l d prior p i tto transfusion t f i has h been b challenging. h ll gi g ‒ A non non-invasive, invasive routine screening test for MP is needed needed. ‒ Factors contributing to MP formation and the role MP play in adverse reactions to transfusion are still unknown unknown. ‒ Currently research testing for MP uses flow cytometry and proteomics. proteomics ‒ Limitations Li it ti off these th techniques t h i are: time ti consuming i sample l preparation, ti invasive, i i flow fl cytometry t t fails f il to t detect d t t MP < 200 nm by b scattering. tt i ‒ ThromboLUX Th b LUX (Fi ((Fig. g 5)) uses th the p principle i ipl off d dynamic y i lilight ght scattering tt i g tto d determine t i th the ki kind d off p particles ti l present p t in i platelet pl t l t components, p t , their th i concentration t ti and d response p to t temperature stress stress. MP are easily detected due to their high speed of motion motion. Objectives j ‒ Determine whether ThromboLUX can be used to screen blood components for MP MP. ‒ Develop a quick routine test method to measure MP concentration in blood components components. E Experimental i t l Methods M th d ‒ ‒ ‒ ‒ ‒ 100 µL of platelet concentrate (PC) or leukoreduced SAGM SAGM-stored stored erythrocyte concentrate (EC) supernatant were loaded into capillaries for ThromboLUX testing testing. C t ifugattio a Centrifugation Ce and d washing ashi g steps t ffor o EC C were e e only o ly required equi ed d for fo co comparative pa atti e flow flo cytometry. cyto t ett y I Th In ThromboLUX b LUX th the D Doppler ppl shift hift off th the lilight ght scattered tt d b by y suspended p d dp particles ti l iis analysed ly d to t determine d t i MP size i and d content. t t Known concentrations of reference particles (platelets (platelets, beads) allow the calculation of MP concentrations by intra intra-sample sample comparison comparison. All samples were analyzed with a FACSCanto II flow cytometer. cytometer Re elaativve e In nte enssityy [%]] R Results lt MP 0.20 0 18 0.18 0.16 Platelets PC Day 1 y 0.14 0.12 0 10 0.10 PC Day 5 PC Day 5 0.08 0.06 ThromboLUX Flow Cytometry Concentration of Reference Particles [ ] [MP] (particles/L) [ ] [MP] (particles/L) 1 1 E 12 1.17E+12 8 81E 12 8.81E+12 1 69E 12 1.69E+12 1.18E+12 8.37E+12 1.82E+12 9.80E+11 6.45E+12 3.66E+12 7.80E+11 7.80E 11 6.14E+08 6 08 1.95E+08 1.95E 08 5 67E+11 5.67E 11 1.74E+10 1.74E 10 1 51E+10 1.51E 10 5 67E+11 5.67E+11 1 82E+11 1.82E+11 1 36E+11 1.36E+11 0.04 0.02 0.00 Hydrodynamic Radius [nm] Hydrodynamic Radius [nm] Fig 1 Fig. 1. ThromboLUX histogram shows a small MP population that is stable during the 5-day 5 day shelf life relative to a distinct platelet population population. MP are primarily determined by the donor . Re elaativve In nte enssityy [%]] Table 1. ThromboLUX concentration estimates of different MP populations in a platelet concentrate (PC, Fig. 1), lipemic platelet-rich platelet rich plasma (PRP, (PRP Fig Fig. 3) and the supernatant of a red blood cell concentrate (RBC (RBC, Fig Fig. 4) 4). Note that EMP b by flflow cytometry t t only l takes t k specifically ifi ll labelled l b ll d EMP iinto t account. t 0 30 0.30 0.25 Li Lipemic i Chylomicrons Description PC D 1 Day Pl l Platelets Platelets Day y5 PC 0.20 0.15 Sample Reference R f Particles Lipid removed p Lipemic p Platelets Lipid p d removed e o ed 0.10 0 05 0.05 RBC 0.00 EMP 10 (1:9 dil) Latex Beads EMP 90 Hydrodynamic Radius [nm] Hydrodynamic Radius [nm] R lattive Rel e Inte en nsitty [% %] Fig 2 Fig. 2. ThromboLUX histogram shows small fat droplets (chylomicrons) next to one distinct platelet population population. 0.07 0.06 0.05 EMP 10 EMP 10 EMP 90 EMP 90 EMP Known Reference Concentration Known Reference Concentration Estimated MP Concentration Estimated MP Concentration Beads 0.04 0.03 0.02 0.01 0.00 Hydrodynamic Radius [nm] yd ody a c ad us [ ] Fig 3 Fig. 3. ThromboLUX histogram shows one distinct population of erythrocyte-derived microparticles i ti l (EMP) relative l ti to t a reference f population l ti off beads. b d Fig 4 Fig. 4. Example of estimation of MP concentration [MP] shown for EMP90 from Fig Fig. 3 3. The concentration of reference particles (in this case beads) is entered with the known peak number. number The size of the unknown peak is calculated. p MP iin platelet pl t l t concentrates t t measured db by y ThromboLUX Th b LUX hi highly ghly correlate l t with ith concentration t ti from f flow fl cytometry yt t y (r ( = 0.983, 0 983, p < 0.005) 0 005)) and d are stable t bl d during i g storage t g (Fi (Fig.1). ( g 1)) Chylomicrons y in lipemic p plasma can be removed byy ultracentrifugation p g and lipid-free lipid p free p plasma can be used to resuspend p platelets ((Fig. p (Fig g 2) 2). ) EMP could be detected in the red blood cell supernatant without the extensive sample preparation but for comparative testing with flow cytometry centrifugation is required (Fig 3) (Fig. 3). It has to be noted that, that in contrast to ThromboLUX, ThromboLUX the flow cytometer counts labeled MP MP. More work is required to determine the correlation correlation. Beads are added if no reference particles are left in the sample. With known reference peak, software automatically estimates [MP] (Fig. 4). Conclusions Conclusions ‒ ThromboLUX can detect platelet derived MP populations in a size range of 20 – 1100 nm and discriminate them from larger particles in the same sample sample. ‒ Minimal Mi i l sample l preparation ti could ld compensate t for f the th lack l k off differentiation diff ti ti which hi h might i ht be b irrelevant i l t att a pre-screening pre screening i stage. t ‒ ThromboLUX Th b LUX h has the th potential p t ti l as a routine, ti , non-invasive i i test t t to t measure MP in i blood bl d components. p t Acknowledgments Fig 5 Fig. 5. ThromboLUX We th W thank k th the bl blood dd donors and d the th collection ll ti staff t ff att NetCAD N tCAD without ith t whom h this thi study t dy would ld nott have h been b possible. p ibl We W acknowledge k l dg the th C Canada d F Foundation d ti ffor IInnovation ti and d th the Mi Michael h lS Smith ith Foundation for Health Research for infrastructure funding at the UBC Centre for Blood Research.
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