Establishment of Reference Values for Platelet Activation Markers by Flow Cytometry

Sin Won Kim; Young Ae Lim

doi:10.3343/kjlm.2006.26.5.323

Journal List > Korean J Lab Med > v.26(5) > 1011336

Go to TopGo to Top Go to BottomGo to Bottom

TOOLS

Kim and Lim: Establishment of Reference Values for Platelet Activation Markers by Flow Cytometry

Original Article

Korean J Leg Med 2006;26(5):323-328.

Published online: 10 February 2006

DOI: https://doi.org/10.3343/kjlm.2006.26.5.323

Establishment of Reference Values for Platelet Activation Markers by Flow Cytometry

Sin Won Kim, M.D., Young Ae Lim, M.D.

Department of Laboratory Medicine, Ajou University School of Medicine, Suwon, Korea

Received 13 July 2006 Revised 4 September 2006 Accepted 17 September 2006

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

This study was purposed to establish reference values for platelet activation markers and leukocyte-platelet aggregates in the evaluation of the platelet function tests using flow cytometry.

Methods

Whole blood samples were obtained from 30 volunteers of healthy adults. Diluted blood samples, either in the resting state or in activated state by the addition of agonist, 20 μM ADP or 100 μM TRAP, were stained with fluorescent conjugated monoclonal antibody of PAC1 or CD62P. Then, the percentages of expression for each marker were analyzed by flow cytometry. For leukocyte-platelet aggregates, monoclonal antibodies of CD41a, CD14 and CD45 were added simultaneously to undiluted whole blood.

Results

Reference values for the percentages of the expression of PAC1 and CD62P, respectively, were 0.1–12.5% and 0.0–4.7% at the resting state, 65.3–92.4% and 39.0–75.7% with the addition of 20 μM ADP, and 68.1–93.1% and 60.5–91.2% with the addition of 100 μM TRAP. Reference values for leukocyte-platelet aggregates, granulocyte-platelet aggregates, and lymphocyte-platelets aggregates were 2.8–23.6%, 5.3–34.2%, and 4.9–21.6%, respectively.

Conclusions

The platelet activation markers at the resting or an activated state with agonists and leukocyte-platelet aggregates could be analyzed using flow cytometry. These reference values should be helpful in interpreting platelet function tests by flow cytometry.

Keywords: Platelet activation, Reference values, Flow cytometry, Leukocyte-platelet

References

1. Flores NA, Sheridan DJ. The pathophysiological role of platelets during myocardial ischaemia. Cardiovasc Res. 1994; 28:295–302.

2. Michelson AD. Flow cytometry: a clinical test of platelet function. Blood. 1996; 87:4925–36.

3. Rinder HM, Bonan JL, Rinder CS, Ault KA, Smith BR. Activated and unactivated platelet adhesion to monocytes and neutrophils. Blood. 1991; 78:1760–9.

4. Harrison P, Cramer EM. Platelet alpha-granules. Blood Rev. 1993; 7:52–62.

5. Shattil SJ, Hoxie JA, Cunningham M, Brass LF. Changes in the platelet membrane glycoprotein IIb-IIIa complex during platelet activation. J Biol Chem. 1985; 260:11107–14.

6. Xiao Z, Theroux P. Clopidogrel inhibits platelet-leukocyte interactions and thrombin receptor agonist peptide-induced platelet activation in patients with an acute coronary syndrome. J Am Coll Cardiol. 2004; 43:1982–8.

7. Yamazaki M, Uchiyama S, Iwata M. Measurement of platelet fibrinogen binding and p-selectin expression by flow cytometry in patients with cerebral infarction. Thromb Res. 2001; 104:197–205.

8. Lim YA, Hyun BH. Evaluation of platelet parameters on the ADVIA 120 as the quality indicator for stored platelets. Clin Lab Haematol. 2002; 24:377–84.

9. Li N, Goodall AH, Hjemdahl P. A sensitive flow cytometric assay for circulating platelet-leucocyte aggregates. Br J Haematol. 1997; 99:808–16.

10. Villmow T, Kemkes-Matthes B, Matzdorff AC. Markers of platelet activation and platelet-leukocyte interaction in patients with myeloproliferative syndromes. Thromb Res. 2002; 108:139–45.

11. Jacoby RC, Owings JT, Holmes J, Battistella FD, Gosselin RC, Paglieroni TG. Platelet activation and function after trauma. J Trauma. 2001; 51:639–47.

12. Holmes MB, Sobel BE, Howard DB, Schneider DJ. Differences between activation thresholds for platelet P-selectin glycoprotein IIb-IIIa expression and their clinical implications. Thromb Res. 1999; 95:75–82.

13. Roshan TM, Normah J, Rehman A, Naing L. Effect of menopause on platelet activation markers determined by flow cytometry. Am J Hematol. 2005; 80:257–61.

14. Michelson AD, Barnard MR, Hechtman HB, MacGregor H, Connolly RJ, Loscalzo J, et al. In vivo tracking of platelets: circulating degranulated platelets rapidly lose surface P-selectin but continue to circulate and function. Proc Natl Acad Sci USA. 1996; 93:11877–82.

15. Hagberg IA, Lyberg T. Evaluation of circulating platelet-leukocyte conjugates: a sensitive flow cytometric assay well suited for clinical studies. Platelets. 2000; 11:151–60.

16. Peters MJ, Heyderman RS, Hatch DJ, Klein NJ. Investigation of platelet-neutrophil interactions in whole blood by flow cytometry. J Immunol Methods. 1997; 209:125–35.

Fig. 1.

Dots plot (A) and histograms (B) for FITC-PAC1 and PE-CD62P platelets after stimulation by 20 μM ADP or 100 μM TRAP. Gated R8 was defined as platelets by forward and side scatter characteristics. Histograms (B) are overlayed with isotype control, and the percentages of PAC1 expressing platelets after stimulation by 20 μM ADP and 100 μM TRAP were 83.1% and 89.1% (M1), and those of CD62P were 80.7% and 83.4% (M1), 29.2% and 68.3 (M2).

Fig. 2.

Flow cytometric analysis of leukocyte-platelet aggregates using 3 color labeling (FITC-CD41a, PE-CD14, PerCP-CD45) in whole blood. The leukocyte populations are defined by their size (forward side scatter, FSC) and anti-CD45 densities (R1). Anti-CD45 and anti-CD14 were used to distinguish neutrophils (R3), lymphocyte (R4), monocytes (R5) and total leukocytes (R2). The histogram represents neutrophilplatelet aggregates as the percentages of CD41a expressed platelets (M1) in neutrophils (R3).

Table 1.

Reference values (central 95 percentile) for percentages of expression and delta expression (Δ) of platelets labeling FITC-PAC1 or PE-CD62P in the resting state or in an activated state with stimulation by 20 μM ADP or 100 μM TRAP

	Resting state	Activated state with stimulation by		Δ between resting state with stimulation by
	Resting state	20 μM ADP	100 μM TRAP	20 μM ADP	100 μM TRAP
PAC1	0.3–12.5	65.3–92.4	68.1–93.1	64.2–89.7	63.6–91.6
CD62P	0.0–4.7	39.0–75.7^*	60.5–91.2	37.6–73.3^*	59.4–90.7

Delta expression (Δ) means a difference in percentage expression between platelets labeling FITC-PAC1 or PE-CD62P in resting state and activated state with stimulation by 20 μM ADP or 100 μM TRAP.

^* : P< 0.01 versus stimulation by 100 μM TRAP.

TOOLS

Similar articles