Whole Blood Analysis of Leukocyte‐Platelet Aggregates

Anja J. Gerrits1, Andrew L. Frelinger1, Alan D. Michelson1

1 Center for Platelet Research Studies, Division of Hematology/Oncology, Boston Children's Hospital, Dana‐Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.15
DOI:  10.1002/cpcy.8
Online Posting Date:  October, 2016
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Abstract

In inflammatory and thrombotic syndromes, platelets aggregate with circulating leukocytes, especially monocytes and neutrophils. This leukocyte‐platelet aggregate formation is initiated primarily through platelet surface expression of P‐selectin (CD62P), following activation‐dependent degranulation of α‐granules, binding to its constitutively expressed counter‐receptor, P‐selectin glycoprotein ligand 1 (PSGL‐1), on leukocytes. Monocyte‐platelet aggregates are a more sensitive marker of platelet activation than platelet surface P‐selectin. Detection of leukocyte‐platelet aggregates is relatively simple by whole‐blood flow cytometry. Light scatter and at least one leukocyte‐specific antibody are used to gate the desired population, and the presence of associated platelets is detected by immunostaining for abundant platelet‐specific markers. © 2016 by John Wiley & Sons, Inc.

Keywords: flow cytometry; leukocytes; platelets; P‐selectin; PSGL‐1; coronary artery disease; activation‐dependent receptors

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Whole Blood Analysis of Leukocyte‐Platelet Aggregates
  • Alternate Protocol 1: Whole Blood Analysis of Leukocyte‐Platelet Aggregates in Prefixed Samples
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Whole Blood Analysis of Leukocyte‐Platelet Aggregates

  Materials
  • Platelet‐specific antibody: monoclonal anti‐CD42a, ‐CD42b, ‐CD41, or ‐CD61 (one or more of these antigens may be missing in rare inherited disorders, e.g., Bernard‐Soulier syndrome and Glanzmann thrombasthenia; see unit 6.10, Krueger et al., )
  • Leukocyte‐specific antibody: monoclonal anti‐CD14, ‐CD64, ‐CD33, or ‐CD45 (this reagent is diluted in an antibody cocktail with the platelet‐specific antibody)
  • Negative control: an isotype‐, fluorophore‐, concentration‐, and F:P ratio‐matched non‐specific antibody for background fluorescence determination (to be used in conjunction with the platelet‐specific antibody)
  • Platelet agonist: such as adenosine diphosphate (ADP; Chrono‐Log, cat. no. 384) or thrombin receptor‐activating peptide (TRAP; Bachem, cat. no. H‐2936; see reciperecipes)
  • Modified HEPES/Tyrode's (HT) buffer (see recipe)
  • Source of human whole blood (patient or donor)
  • Fix/lyse solution: any red‐cell lysis reagent optimized for flow cytometry and containing formaldehyde, e.g., FACS lysing solution, Becton Dickinson, or Optilyse, Beckman Coulter (the performance of these products must be assessed by the individual laboratory)
  • Blood collection tubes containing buffered sodium citrate or other suitable anticoagulant (BD Vacutainers) (see unit 6.10, Krueger et al., )
  • 21‐G (or larger bore) needles
  • Flow cytometer with 488‐nm excitation and suitable filters for collection of fluorophore emissions
NOTE: Individual laboratories must determine optimal assay conditions and sample stability.

Alternate Protocol 1: Whole Blood Analysis of Leukocyte‐Platelet Aggregates in Prefixed Samples

  Additional Materials (also see protocol 1Basic Protocol)
  • 5‐ml tubes
  • Centrifuge
  • Vortex mixer
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Figures

Videos

Literature Cited

Literature Cited
  Berny‐Lang, M.A., Frelinger, A.L. III, Barnard, M.R., and Michelson, A.D. 2013. Flow cytometry. In Platelets, 3rd ed. (A.D. Michelson, ed) pp.581‐602. Academic Press/Elsevier Science, New York.
  Bledzka, K., Pesho, M.M., Ma, Y., and Plow, E.F. 2013. Integrin alphaIIb‐beta3. In Platelets, 3rd ed. (A.D. Michelson, ed) pp. 233‐248. Academic Press/Elsevier Science, New York.
  Bruijne‐Admiraal, L.G., Modderman, P.W., von dem Borne, A.E., and Sonnenberg, A. 1992. P‐selectin mediates Ca(2+)‐dependent adhesion of activated platelets to many different types of leukocytes: Detection by flow cytometry. Blood 80:134‐142. doi: http://dx.doi.org/
  Chandraratne, S., von Bruehl, M.L., Pagel, J.I., Stark, K., Kleinert, E., Konrad, I., Farschtschi, S., Coletti, R., Gärtner, F., Chillo, O., Legate, K.R., Lorenz, M., Rutkowski, S., Caballero‐Martinez, A., Starke, R., Tirniceriu, A., Pauleikhoff, L., Fischer, S., Assmann, G., Mueller‐Hoecker, J., Ware, J., Nieswandt, B., Schaper, W., Schulz, C., Deindl, E., and Massberg, S. 2015. Critical role of platelet glycoprotein ibα in arterial remodeling. Arterioscler. Thromb. Vasc. Biol. 35:589‐597. doi: 10.1161/ATVBAHA.114.304447.
  Diacovo, T.G., Roth, S.J., Buccola, J.M., Bainton, D.F., and Springer, T.A. 1996. Neutrophil rolling, arrest, and transmigration across activated, surface‐adherent platelets via sequential action of P‐selectin and the beta 2‐integrin CD11b/CD18. Blood 88:146‐157.
  Elalamy, I., Chakroun, T., Gerotziafas, G.T., Petropoulou, A., Robert, F., Karroum, A., Elgrably, F., Samama, M.M., and Hatmi, M. 2008. Circulating platelet‐leukocyte aggregates: A marker of microvascular injury in diabetic patients. Thromb. Res. 121:843‐848. doi: 10.1016/j.thromres.2007.07.016.
  Evangelista, V. and Smyth, S.S. 2013. Interactions between platelets, leukocytes, and the endothelium. In Platelets, 3rd ed. (A.D. Michelson, ed.) pp. 295‐312. Academic Press/Elsevier Science, New York.
  Ferroni, P., Riondino, S., Vazzana, N., Santoro, N., Guadagni, F., and Davi, G. 2012. Biomarkers of platelet activation in acute coronary syndromes. Thromb. Haemost. 108:1109‐1123. doi: 10.1160/TH12‐08‐0550.
  Furman, M.I., Benoit, S.E., Barnard, M.R., Valeri, C.R., Borbone, M.L., Becker, R.C., Hechtman, H.B., and Michelson, A.D. 1998. Increased platelet reactivity and circulating monocyte‐platelet aggregates in patients with stable coronary artery disease. J. Am. Coll. Cardiol. 31:352‐358. doi: 10.1016/S0735‐1097(97)00510‐X.
  Furman, M.I., Barnard, M.R., Krueger, L.A., Fox, M.L., Shilale, E.A., Lessard, D.M., Marchese, P.J., Frelinger, A.L. III, Goldberg, R., and Michelson, A.D. 2001. Circulating monocyte‐platelet aggregates are an early marker of acute myocardial infarction. J. Am. Coll. Cardiol. 38:1002‐1006. doi: 10.1016/S0735‐1097(01)01485‐1.
  Gardiner, E.E., De Luca, M., McNally, T., Michelson, A.D., Andrews, R.K., and Berndt, M.C. 2001. Regulation of P‐selectin binding to the neutrophil P‐selectin counter‐receptor P‐selectin glycoprotein ligand‐1 by neutrophil elastase and cathepsing G. Blood 98:1440‐1447. doi: 10.1182/blood.V98.5.1440.
  George, J.N., Pickett, E.B., Saucerman, S., McEver, R.P., Kunicki, T.J., Kieffer, N., and Newman, P.J. 1986. Platelet surface glycoproteins. Studies on resting and activated platelets and platelet membrane microparticles in normal subjects, and observations in patients during adult respiratory distress syndrome and cardiac surgery. J. Clin. Invest. 78: 340‐348. doi: 10.1172/JCI112582.
  Goncalves, R., Zhang, X., Cohen, H., Debrabant, A., and Mosser, D.M. 2011. Platelet activation attracts a subpopulation of effector monocytes to sites of Leishmania major infection. J. Exp. Med. 208:1253‐1265. doi: 10.1084/jem.20101751.
  Hara, T., Shimizu, K., Ogawa, F., Yanaba, K., Iwata, Y., Muroi, E., Takenaka, M., Komura, K., Hasegawa, M., Fujimoto, M., and Sato, S. 2010. Platelets control leukocyte recruitment in a murine model of cutaneous arthus reaction. Am. J. Pathol. 176:259‐269. doi: 10.2353/ajpath.2010.081117.
  Harding, S.A., Sommerfield, A.J., Sarma, J., Twomey, P.J., Newby, D.E., Frier, B.M., and Fox, K.A. 2004. Increased CD40 ligand and platelet‐monocyte aggregates in patients with type 1 diabetes mellitus. Atherosclerosis 176:321‐325. doi: 10.1016/j.atherosclerosis.2004.05.008.
  Harding, S.A., Din, J.N., Sarma, J., Jessop, A., Weatherall, M., Fox, K.A., and Newby, D.E. 2007. Flow cytometric analysis of circulating platelet‐monocyte aggregates in whole blood: Methodological considerations. Thromb. Haemost. 89:451‐456.
  Hui, H., Fuller, K., Erber, W.N., and Linden M.D. 2015. Measurement of monocyte‐platelet aggregates by imaging flow cytometry. Cytometry A 87A:273‐278. doi: 10.1002/cyto.a.22587.
  Joseph, J.E., Harrison, P., Mackie, I.J., Isenberg, D.A., and Machin, S.J. 2001. Increased circulating platelet‐leucocyte complexes and platelet activation in patients with antiphospholipid syndrome, systemic lupus erythematosus and rheumatoid arthritis. Br. J. Haematol. 115:451‐459. doi: 10.1046/j.1365‐2141.2001.03101.x.
  Krueger, L. A., Barnard, M. R., Frelinger, A., Furman, M. I. and Michelson, A. D. 2002. Immunophenotypic Analysis of Platelets. Curr. Protoc. Cytom. 19:6.10:6.10.1‐6.10.17.
  LaRosa, C.A., Rohrer, M.J., Benoit, S.E., Barnard, M.R., and Michelson, A.D. 1994. Neutrophil cathepsin G modulates the platelet surface expression of the glycoprotein (GP) Ib‐IX complex by proteolysis of the von Willebrand factor binding site on GPIba and by a cytoskeletal‐mediated redistribution of the remainder of the complex. Blood 84:158‐168.
  Linden, M.D. and Jackson, D.E. 2010. Platelets: Pleiotropic roles in atherogenesis and atherothrombosis. Int. J. Biochem. Cell Biol. 42:1762‐1766. doi: 10.1016/j.biocel.2010.07.012.
  Majumder, B., North, J., Mavroudis, C., Rakhit, R., and Lowdell, M.W. 2012. Improved accuracy and reproducibility of enumeration of platelet‐monocyte complexes through use of doublet‐discriminator strategy. Cytometry B 82B:353‐359. doi: 10.1002/cyto.b.21040.
  Mauler, M., Seyfert, J., Haenel, D., Seeba, H., Guenther, J., Stallmann, D., Schoenichen, C., Hilgendorf, I., Bode, C., Ahrens, I., and Duerschmied D. 2016. Platelet‐neutrophil complex formation‐a detailed in vitro analysis of murine and human blood samples. J. Leukoc. Biol. 99:781‐789. doi: 10.1189/jlb.3TA0315‐082R.
  Michelson, A.D. 1994. Platelet activation by thrombin can be directly measured in whole blood through the use of the peptide GPRP and flow cytometry: Methods and clinical studies. Blood Coagul. Fibrinolysis 5:121‐131. doi: 10.1097/00001721‐199402000‐00014.
  Michelson, A.D., Barnard, M.R., Krueger, L.A., Valeri, C.R., and Furman, M.I. 2001. Circulating monocyte‐platelet aggregates are a more sensitive marker of in vivo platelet activation than platelet surface P‐selectin: Studies in baboons, human coronary intervention, and human acute myocardial infarction. Circulation 104:1533‐1537. doi: 10.1161/hc3801.095588.
  Mickelson, J.K., Lakkis, N.M., Villarreal‐Levy, G., Hughes, B.J., and Smith, C.W. 1996. Leukocyte activation with platelet adhesion after coronary angioplasty: A mechanism for recurrent disease? J. Am. Coll. Cardiol. 28:345‐353. doi: 10.1016/0735‐1097(96)00164‐7.
  Montoro‐Garcia, S., Shantsila, E., Hernández‐Romero, D., Jover, E., Valdés, M., Marin, F., and Lip, G.Y. 2014. Small‐size platelet microparticles trigger platelet and monocyte functionality and modulate thrombogenesis via P‐selectin. Br. J. Haematol. 166:571‐580. doi: 10.1111/bjh.12913.
  Nieuwland, R., van der Pol, E., Gardiner, C., and Sturk, A. 2013. Platelet‐derived microparticles. In Platelets, 3rd ed. (A.D. Michelson, ed.) pp. 453‐467. Academic Press/Elsevier Science, New York.
  Ott, I., Neumann, F.J., Gawaz, M., Schmitt, M., and Schomig, A. 1996. Increased neutrophil‐platelet adhesion in patients with unstable angina. Circulation 94:1239‐1246. doi: 10.1161/01.CIR.94.6.1239.
  Palabrica, T., Lobb, R., Furie, B.C., Aronovitz, M., Benjamin, C., Hsu, Y.M., Sajer, S.A., and Furie, B. 1992. Leukocyte accumulation promoting fibrin deposition is mediated in vivo by P‐selectin on adherent platelets. Nature 359:848‐851. doi: 10.1038/359848a0.
  Parimon, T., Li, Z., Bolz, D.D., McIndoo, E.R., Bayer, C.R., Stevens, D.L., and Bryant, A.E. 2013. Staphylococcus aureus a‐hemolysin promotes platelet‐neutrophil aggregate formation. J. Infect. Dis. 208:761‐770. doi: 10.1093/infdis/jit235.
  Powell, C.C., Rohrer, M.J., Barnard, M.R., Peyton, B.D., Furman, M.I., and Michelson, A.D. 1999. Chronic venous insufficiency is associated with increased platelet and monocyte activation and aggregation. J. Vasc. Surg. 30:844‐851. doi: 10.1016/S0741‐5214(99)70009‐1.
  Rinder, C.S., Bonan, J.L., Rinder, H.M., Mathew, J., Hines, R., and Smith, B.R. 1992. Cardiopulmonary bypass induces leukocyte‐platelet adhesion. Blood 79:1201‐1205. doi: http://dx.doi.org/
  Rutten, B., Roest, M., McClellan, E.A., Sels, J.W., Stubbs, A., Jukema, J.W., Doevendans, P.A., Waltenberger, J., van Zonneveld, A.J., Pasterkamp, G., De Groot, P.G., and Hoefer, I.E. 2016. Platelet density per monocyte predicts adverse events in patients after percutaneous coronary intervention. Thromb. Haemost. 115:353‐360. doi: 10.1160/TH15‐03‐0227.
  Seizer, P., Gawaz, M., and May A.E. 2008. Platelet‐monocyte interactions – A dangerous liaison linking thrombosis, inflammation and atherosclerosis. Curr. Med. Chem. 15:1976‐1980. doi: 10.2174/092986708785132852.
  Simon, D.I., Chen, Z., Xu, H., Li, C.Q., Dong, J., McIntire, L.V., Ballantyne, C.M., Zhang, L., Furman, M.I., Berndt, M.C., and Lopez, J.A. 2000. Platelet glycoprotein Ibα is a counterreceptor for the leukocyte integrin Mac‐1 (CD11b/CD18). J. Exp. Med. 192:193‐204. doi: 10.1084/jem.192.2.193.
  Tekelioğlu, Y. and Uzun, H. 2013. Circulating platelet‐leukocyte aggregates in patients with inflammatory bowel disease. J. Chin. Med. Assoc. 76:182‐185. doi: 10.1016/j.jcma.2012.12.015.
  Yip, C., Ignjatovic, V., Attard, C., Monagie, P., and Linden, M.D. 2013. First report of elevated monocyte‐platelet aggregates in healthy children. PLoS ONE 8:e67416. doi: 10.1371/journal.pone.0067416.
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