Variation in activation marker expression within the platelet population – a new parameter for evaluation of platelet flow cytometry data

Figures & data

Figure 1. The potential and limitations of the different ways to report platelet activation responses, the percentage positive platelets (“%Gated,” red boxes), median fluorescence intensity (“X-Med,” blue boxes) and variation of the fluorescence response (“X-CV,” Orange boxes), as illustrated with (A) a sample with low activation (resting platelets, P-selectin as activation marker), (B) a sample with moderately activated platelets (fibrinogen binding as activation marker) and (C) a sample with strongly activated platelets (P-selectin as activation marker). Results for “All” refers to all particles within the “All platelets” gate shown in Figure 2.

Figure 2. The gating strategy used for analysis of the PCs in this study. PE-positive particles were collected using a threshold on FL2 at acquisition. The single platelets were identified based on FS height and area (A) and then distinguished from platelet fragments through PE fluorescence (B) with a platelet gate big enough to include also platelets with a change in forward scatter due to activation and procoagulant transformation. The setting of the gate for P-selectin positive platelets as close as possible to 1.5% with the use of the negative (isotype antibody) control sample (C). Data obtained from an activated sample containing P-selectin antibody when the above presented gating strategy was used (D).

Table 1. Median, confidence interval (CI) and p-value for apheresis and IPU with all tested agonists and markers. Significant results are marked in bold.

Agonist (final concentration)	Activation marker, investigated parameter	Apheresis PCs (n = 13)	IPU PCs (n = 26)	P-value	Significance
HEPES buffer (spontaneous activation)	Fibrinogen binding, % pos	4.1 (3.4–4.4)	4.2 (3.6–4.5)	0.6011	No
	Fibrinogen binding, MFI	0.54 (0.48–0.57)	0.60 (0.59–0.67)	0.0006	Yes
	Fibrinogen binding, CV	241 (159–334)	133 (118–159)	0.0250	Yes
	P-selectin exposure, % pos	48.6 (42.5–61.8)	64.5 (61.1–68.6)	0.0001	Yes
	P-selectin exposure, MFI	1.43 (1.10–2.05)	2.17 (1.86–2.37)	0.0010	Yes
	P-selectin exposure, CV	231 (192–269)	189 (180–202)	0.0005	Yes
	LAMP-1 exposure, % pos	8.4 (6.2–9.1)	8.1 (6.7–8.6)	0.8083	No
	LAMP-1 exposure, MFI	0.20 (0.18–0.20)	0.19 (0.18–0.19)	0.3002	No
	LAMP-1 exposure, CV	60 (53–98)	62 (58–67)	0.4008	No
	GPIb expression, MFI	24.37 (21.28–28.49)	23.40 (21.90–25.24)	0.4484	No
	GPIb expression, CV	44 (40–47)	49 (48–54)	<0.0001	Yes
PAR1-AP (10 µM)	Fibrinogen binding, % pos	30.7 (22.8–45.8)	20.1 (14.6–27.0)	0.0022	Yes
	Fibrinogen binding, MFI	1.82 (1.46–1.98)	1.37 (1.12–1.51)	0.0002	Yes
	Fibrinogen binding, CV	113 (93–199)	102 (97–114)	0.4525	No
	P-selectin exposure, % pos	98.3 (97.0–99.0)	96.6 (95.8–97.4)	0.0001	Yes
	P-selectin exposure, MFI	26.83 (23.44–30.26)	21.65 (19.41–24.37)	0.0009	Yes
	P-selectin exposure, CV	74 (66–85)	84 (78–87)	0.0020	Yes
	CD63 exposure, % pos	84.5 (81.7–87.8)	74.25 (67.20–78.70)	<0.0001	Yes
	CD63 exposure, MFI	1.00 (0.88–1.10)	0.62 (0.51–0.74)	<0.0001	Yes
	CD63 exposure, CV	85 (79–152)	112 (105–156)	0.0009	Yes
PAR4-AP (100 µM)	Fibrinogen binding, % pos	33.7 (23.3–50.7)	19.8 (13.8–23.3)	0.0007	Yes
	Fibrinogen binding, MFI	1.77 (1.63–2.20)	1.20 (1.10–1.57)	<0.0001	Yes
	Fibrinogen binding, CV	96 (69–118)	95 (89–98)	0.7353	No
	P-selectin exposure, % pos	98.0 (97.0–99.0)	95.0 (94.0–96.0)	<0.0001	Yes
	P-selectin exposure, MFI	24.54 (20.11–26.34)	14.81 (12.87–15.88)	<0.0001	Yes
	P-selectin exposure, CV	73 (70–77)	91 (86–96)	0.0003	Yes
	LAMP-1 exposure, % pos	48 (38–51)	19 (15–24)	<0.0001	Yes
	LAMP-1 exposure, MFI	0.35 (0.30–0.37)	0.21 (0.20–0.23)	<0.0001	Yes
	LAMP-1 exposure, CV	66 (64–72)	70 (67–76)	0.1188	No
CRP-XL (0.15 µg/mL)	Fibrinogen binding, % pos	61.9 (41.6–69.1)	41.3 (37.2–50.0)	0.0063	Yes
	Fibrinogen binding, MFI	2.89 (2.27–3.49)	2.18 (1.86–2.63)	0.0288	Yes
	Fibrinogen binding, CV	97 (80–131)	101 (91–109)	0.8485	No
	P-selectin exposure, % pos	96.5 (92.7–97.7)	94.8 (93.3–96.4)	0.2220	No
	P-selectin exposure, MFI	24.92 (18.18–27.68)	20.93 (17.80–24.20)	0.2944	No
	P-selectin exposure, CV	81 (75–97)	89 (83–97)	0.1117	No
	LAMP-1 exposure, % pos	44.0 (31.7–57.0)	33.2 (28.2–35.8)	0.0084	Yes
	LAMP-1 exposure, MFI	0.33 (0.25–0.39)	0.25 (0.24–0.29)	0.0064	Yes
	LAMP-1 exposure, CV	71 (69–74)	77 (73–81)	0.0313	Yes

Results are presented as median and 95% CI within parenthesis for PCs stored for 6–7 days. Significant difference between apheresis PCs and IPU PCs was considered when the p-value was <0.05 (Mann-Whitney test). PC = platelet concentrate, % pos = percentage positive platelets, MFI = median fluorescence intensity, CV = coefficient of variation, PAR1-AP = PAR1-activating peptide, PAR4-AP = PAR4-activating peptide, CRP-XL = cross-linked collagen-related peptide.

Table 2. Impact of the number of platelet events investigated on the flow cytometry variables.

Activation marker, investigated parameter	Platelet events investigated	PC A	PC B	PC C	PC D
Fibrinogen binding, % pos	5000	42.2	10.7	33.2	19.0
	10 000	43.8	11.4	33.5	20.4
	20 000	44.4	11.8	34.6	22.0
	50 000	45.5	12.9	35.9	22.9
Fibrinogen binding, MFI	5000	1.2	0.7	1.1	0.8
	10 000	1.3	0.7	1.1	0.8
	20 000	1.3	0.7	1.2	0.9
	50 000	1.3	0.8	1.2	0.9
Fibrinogen binding, CV	5000	125	102	370	395
	10 000	126	85	384	382
	20 000	128	120	372	402
	50 000	133	151	381	420
P-selectin exposure, % pos	5000	69.7	17.3	67.5	59.9
	10 000	71.9	16.3	68.5	61.0
	20 000	71.5	17.7	69.6	62.3
	50 000	71.5	17.2	69.6	62.4
P-selectin exposure, MFI	5000	4.2	0.8	3.7	2.7
	10 000	4.6	0.8	3.6	2.7
	20 000	4.7	0.8	3.9	2.8
	50 000	4.5	0.8	3.9	2.8
P-selectin exposure, CV	5000	128	172	138	143
	10 000	123	158	130	150
	20 000	127	172	132	140
	50 000	124	177	132	141
LAMP-1 exposure, % pos	5000	15.3	5.1	11.9	4.7
	10 000	16.0	4.9	11.6	4.9
	20 000	15.8	5.5	12.6	5.4
	50 000	16.5	5.1	13.2	5.5
LAMP-1 exposure, MFI	5000	0.6	0.5	0.6	0.5
	10 000	0.6	0.5	0.6	0.5
	20 000	0.6	0.5	0.6	0.5
	50 000	0.6	0.5	0.6	0.5
LAMP-1 exposure, CV	5000	75	43	56	52
	10 000	59	64	78	55
	20 000	431	42	76	56
	50 000	89	50	62	55

Results from 4 separate PCs investigated (A-D). PC = platelet concentrate, % pos = percentage positive platelets, MFI = median fluorescence intensity, CV = coefficient of variation.

Data availability statement

The raw data used for this publication can be provided by the authors upon reasonable request.