Circulating microparticles, protein C, free protein S and endothelial vascular markers in children with sickle cell anaemia

Figures & data

Fig. 1.  Microparticles (MP) are gated on their size and ability to express phosphatidylserine when stained by Annexin V in the presence of calcium. Platelets (pink) show no expression of phosphatidylserine after staining by AV conjugated with APC, while MP (blue) show >95% positivity.

Fig. 2.  Blood values (total Hb, HbS percentage Hct, MCV) in the different subgroups (control with HbAA, crises, steady state, HbSC, on treatment with hydroxyurea and transfusion).

Fig. 3.  Markers of hemolysis in all subgroups: reticulocytes, LDH and total-bilirubin.

Fig. 4.  Values of WCC, N and PLT in the different subgroups.

Table I. Hemoglobin levels, red cell indices, white cell count (WCC), platelet counts, bilirubin, lactate dehydrogenase (LDH) and reticulocyte counts

	HbAA			Crisis			HbSS			HU treated			Transfusion			HbSC
	n	Median	IR	n	Median	IR	n	Median	IR	n	Median	IR	n	Median	IR	n	Median	IR
Hb (g dl^−1)	14	110.00	106–121	14	71.00	61–81.5	51	83.00	74.8–93.8	29	102.00	99.5–109	15	100.00	92.8–107.8	13	111.00	101.5–113.5
HbF (%)	n/a	n/a	n/a	14	15.25	5.6–17.6	31	16.20	7.0–27.7	30	27.70	19.1–32.0	15	5.00	2.6–7.9	12	4.50	2.8–5.4
HbS (%)	n/a	n/a	n/a	13	76.90	72.2–86.7	31	74.90	67.1–85.8	30	63.65	59.6–70.3	15	25.90	16.2–35.4	12	44.70	43.9–45.5
MCV (fl)	15	77	72.7–81.0	14	77.80	73.1–81.3	53	80.80	72.8–85.8	30	92.90	89.7–102.8	16	87.10	83.8–90.5	13	70.60	65.1–74.6
MCH (pg/cell)	15	27	24.6–28.1	14	25.20	24.3–27.9	53	27.10	24.9–29.6	29	34.90	31.1–36.8	16	30.00	29.3–30.98	13	24.30	22.9–26.6
Bilirubin (µmol L^−1)	8	11.00	6.0–14.5	11	30.00	21.0–41.0	48	31.50	19.0–43.0	27	19.00	15.0–27.0	15	27.00	18.5–43.0	11	15.00	6.0–21.0
LDH (IU L^−1)	8	397.50	384–571	12	1083.00	866–1,719	45	1031.00	783–1,388	26	731.00	664–894	15	780.00	649–1,075	11	497.00	420–562
Neutrophil (×10^9 L^−1)	17	2.40	1.80–3.25	15	9.40	5.70–12.4	51	4.50	3.25–6.83	30	3.00	2.20–3.93	15	4.60	3.40–7.65	13	2.90	2.35–3.50
Platelet (×10^9 L^−1)	17	320.00	290–402	14	249.50	148–362	51	340.00	282–418	29	245.00	177–418	15	281.00	177–331	13	267.00	230–366
WCC (×10^9 L^−1)	17	7.25	5.40–8.98	15	16.00	12.9–19.3	51	13.35	8.67–15.7	30	7.90	6.15–9.63	15	11.40	8.1–14.3	13	8.50	6.20–10.7
Reticulocytes (total µl^−1)	14	49.25	40.5–61.1	13	251.00	105–328	49	285.00	230–348	29	131.00	90.8–164	15	225.00	128–258	13	102.00	76.6–118

IR=interquartile range.

Fig. 5.  MP numbers and percentages.

Fig. 6.  ADM and ET-1, NO and ADM crises by type.

Fig. 7.  NO, ET-1 and ADM subdivided by crises type (chest crises versus other).

Table II. Statistical analysis of indicators of efficacy

	Comparison	p		Comparison	p
HbS (%)	Crisis vs. HU	p<0.001	Bilirubin (mmol L^−1)	Crisis vs. HbAA	p<0.01
	Crisis vs. TXN	p<0.001		Steady state vs. HbAA	p<0.001
	Crisis vs. HbSC	p<0.001		TXN vs. HbAA	p<0.01
	Steady state vs. HU	p<0.01	LDH (IU L^−1)	Crisis vs. HU	p<0.001
	Steady state vs. TXN	p<0.001		Crisis vs. TXN	p<0.05
	Steady state vs. HbSc	p<0.001		Crisis vs. HbSc	p<0.001
	HU vs. TXN	p<0.001		Crisis vs. HbAA	p<0.001
	HU vs. HbSC	p<0.001	WCC (×109 L^−1)	Crisis vs. HbAA	p<0.001
	TXN vs. HbSC	p<0.001		Crisis vs. HU	p<0.001
Hb (g dl^−1)	Crisis vs. HbAA	p<0.001		Steady state vs. HU	p<0.001
	Crisis vs. HU	p<0.001		HbAA vs. steady state	p<0.001
	Crisis vs. steady state	p<0.01	Neutrophil (×109 L^−1)	Crisis vs. steady state	p<0.05
	Crisis vs. TXN	p<0.001		Crisis vs. HU	p<0.001
	Crisis vs. HbSC	p<0.001		Crisis vs. HbAA	p<0.001
	Steady state vs. TXN	p<0.001	Reticulocytes (ml^−1)	Crisis vs. HU	p<0.05
	Steady state vs. HbSC	p<0.001		Crisis vs. HbAA	p<0.001
	Steady state vs. HbAA	p<0.001		Steady state vs. HU	p<0.001
	HU vs. steady state	p<0.001		Steady state vs. HbAA	p<0.001
	TXN vs. normal	p<0.05		TXN vs. HbAA	p<0.001
HbF (%)	Crisis vs. HU	p<0.01
	HU vs. steady state	p<0.05
	HU vs. TXN	p<0.001

Table III. Adrenomedullin (ADM), endothelin-1 (ET-1) and nitric oxide (NO) levels in SCA patients and controls

	HbAA			Crisis			HbSS			HbSC			HU treated			Transfusion
	n	Median	IR	n	Median	IR	n	Median	IR	n	Median	IR	n	Median	IR	n	Median	IR
ADM (nmol L^−1)	17	0.28	0.27–0.31	13	0.19–0.42	0.15	55	0.27	0.25–0.32	13	0.26	0.25–0.32	29	0.30	0.26–0.36	16	0.30	0.25–0.37
ET-1 (pmol L^−1)	17	55.1	45.2–63.9	13	40.5	9.3–46.3	55	50.1	28.4–57.3	13	58.8	28.1–76.5	29	43.6	12.6–49.6	16	42.6	21.7–48.9
NO (µmol L^−1)	14	20.6	15.6–27.9	11	19.4	11.6–22.5	50	22.6	16.9–28.2	11	30.6	20.8–39.5	27	22.2	18.3–28.4	16	19.5	13.4–21.8

IR = interquartile range.

Fig. A1.  From the top to the bottom: staining for AV, for CD61 and for AV & CD61 (PMP). Gating area using the methodology by Shet et al. (16) (red, right) and this by Biró et al. (23) (black, left). No clear demarcation is seen between MP and noise.

Shet AS, Aras O, Gupta K, Hass MJ, Rausch DJ, Saba N et al. Sickle blood contains tissue factor-positive microparticles derived from endothelial cells and monocytes. Blood. 2003; 102: 2678–83.

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Biró E, Sturk-Maquelin KN, Vogel GM, Meuleman DG, Smit MJ, Hack CE et al. Human cell-derived microparticles promote thrombus formation in vivo in a tissue factor-dependent manner. J Thromb Haemost. 2003; 1: 2561–8.

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