Gravity sedimentation reveals functionally and morphologically different platelets in human blood

Figures & data

Figure 1. Schematics of the gravity sedimentation technique. Platelet (PLT) antisedimentation rate (PAR) is measured by comparing the platelet count in the upper and lower fractions of the blood column after 1 h of gravity sedimentation at room temperature. PAR (%) is calculated as (Upper − Lower)/(Upper + Lower) × 100.

Table I. Bulk characteristics and aggregability of platelets after 1-h gravity sedimentation.

	Whole blood	Upper fraction	Lower fraction	p-value
Characteristics
PLT count [G/L]	239 ± 76	285 ± 132	85 ± 33	.003
MPV [fL]	10.4 ± 1.2	1.3 ± 0.6	10.2 ± 0.6	.644
IPF (abs)	7.6 ± 5.3	8.8 ± 4.4	3.5 ± 2.2	.003
H-IPF [%]	1.4 ± 0.8	1.5 ± 1.0	1.3 ± 0.9	.021
LCR	26.9 ± 5.1	27.5 ± 4.0	26.9 ± 4.9	1.000
PAR	50.84 ± 18.76	NA	NA	NA
Impedance aggregometry
AUC	69.9 ± 13.1	103.3 ± 35.8	26.1 ± 9.6	<.001
AUC/PLT count	0.23 ± 0.13^a	0.28 ± 0.15b	0.26 ± 0.12	<.001^ab
AU	130.9 ± 23.7	174.3 ± 53.0	52.2 ± 16.2	<.001
velocity	15.5 ± 3.8	26.5 ± 1.9	6.5 ± 1.7	<.001

PLT count, platelet count; MPV, mean platelet volume; IPF, immature platelet fraction; H-IPF, high fluorescent immature platelet fraction; LCR, platelet large cell ratio; PAR, platelet antisedimentation rate; AUC (arbitrary unit × time), the area under the curve. AU, arbitrary unit indicates the aggregation of platelets; velocity, the slope of the aggregation curve; p-value (<0.05) indicates the difference between upper and lower fractions. Data are presented as mean ± SD. Student’s t-test was used for comparing PLT, MPV, IPF, H-IPF, and LCR. Wilcoxon Signed Rank test was used for comparing AUC, aggregation, and velocity.

Figure 2. Platelet sedimentation kinetics and its association with ESR. Cell counts from three equal fractions (upper, middle, and lower respectively) of the sedimented blood which were separated at five different time points (30, 45 min, and 1, 2, 3 h). (A) Platelet count as a function of time in a subject with physiological ESR; (B) platelet count as a function of time in a subject with high ESR; (C) PAR as a function of we in a cohort of 21 subjects. The data were fit with a monoexponential function with a plateau value of 78.113 (±6.94 SD) and a decay constant of 5.0245 (±2.42 SD).

Figure 3. ADP-induced platelet aggregation. A–C depicts representative platelet aggregometry curves obtained on whole blood (A), the upper (B) and the lower (C) samples, to demonstrate the curve(s) of the bedside multiplate readings (all measurements provided in duplicate). Red and blue traces refer to two independent measurements. Vertical axis: AU stands for arbitrary unit. Horizontal axis: time (sec). Initial velocity (D) and area under the curve (AUC = arbitrary unit × time) (E) measured by whole blood impedance aggregometry in whole blood from healthy subjects (n = 21) and the upper and lower fractions separated after 1-h gravity sedimentation. Data are shown as median and 25th−75th percentiles (Wilcoxon signed rank test). The horizontal dotted lines mark the normal range of AUC given by the manufacturer.

Figure 4. Electron microscopy of platelets. TEM images of platelets of healthy subjects from the lower (A and C) and upper (B and D) fractions. The arrow in C and the asterisk in D point at an alpha-granule and a dense granule, respectively.

Figure 5. Atomic force microscopy of platelets. Amplitude-contrast AFM images of mica-adsorbed washed platelets from the lower (A) and upper (B) fractions. Scale bar 2 µm. Insets show height-contrast AFM images of representative platelets. Scale bar 0.5 µm. Comparison of the volume (C) and central height (D) of the platelets from the different fractions.

Table II. Transmission electron microscopy data of platelets after one-hour gravity sedimentation.

	Lower fraction	Upper fraction	p-value
Area of platelets [nm^2]	2.26 × 10^6 ± 6.3 × 10^5	3.69 × 106 ± 9.3 × 105	<.001
Perimeter of platelets [nm]	7.21 × 10^3 ± 1.55 × 10^3	9.17 × 103 ± 1.42 × 103	<.001

The area and perimeter of platelets were measured with the iTEM program (Olympus Soft Imaging Solutions, Munster, Germany). Data were obtained by measuring the area and perimeter of 50 platelets from each sample. Data of upper and lower fraction were compared. The value of p was calculated with Student’s t-test.

Data availability statement

The data presented in this study are available on request from the corresponding author.