Deformability and fragility studies of RBCs.

<p>(A) RBC deformability and relaxation capacity was studied with a microfluidic device [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125580#pone.0125580.ref012" target="_blank">12</a>]. Scale bar 10 μm. (B) Deformability and fragility were also measured with a spleen-mimicking device [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125580#pone.0125580.ref013" target="_blank">13</a>]. The cells of interest were stained with CFSE, mixed with RBCs of a control donor, and passed through a bed of metal beads [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125580#pone.0125580.ref013" target="_blank">13</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125580#pone.0125580.ref014" target="_blank">14</a>]. Retention was measured by the number of cells that did not pass through the bead layer. The percentages of CFSE-positive cells in the retained and downstream sample were compared to that in the upstream sample (set at 5%, depicted as a dotted line in panel B).</p

Pedigree and RBC morphology of the subjects in this study.

<p>(A) Patients who are clinically diagnosed with PKAN are indicated in black, the healthy relatives are indicated in white. (B) Representative pictures of blood films used for classification of cell shape. (C) Cells are classified in discocyte, echinocyte, acanthocyte or otherwise misshapen. The graph depicts the percentages of different cell morphologies in PKAN patients (marked with an asterisk) and their unaffected family members, compared to healthy donors (control 1+2) (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125580#pone.0125580.s001" target="_blank">S1 Fig</a> for other PKAN families).</p

Measurements of fetal haemoglobin (HbF) in red cells of PKAN patients and their relatives.

<p>Black, patients; grey, controls (Ctrls) and family members. The control value is the mean of 6 control samples. The error bar depicts the standard deviation.</p

Microparticle formation by PKAN RBCs as characterized by flow cytometry.

<p>(A) The amount of RBC MPs in plasma. (B) Membrane composition of in vitro generated MPs. Band 3 and glycophorin A (GpA) was measured on MPs collected after 4 and 24h incubation of the RBCs in Ringer solution at 37 °C.</p

Impaired drug-induced endovesiculation in erythrocytes of patients with acanthocytosis.

<p>Erythrocytes from patients (ChAc, PKAN+, PKAN-) and control donors were subjected to drug-induced endovesiculation using 3 mM primaquine. The amount of FITC-dextran positive cells (in %) was assessed by flow cytometry as described. Respective pairs of patient and control donors are connected by lines. The data of the MPAN patient within the NBIA/PKAN- cohort is shown as a dashed line.</p

Dose-dependent uptake of fluid phase FITC-dextran by erythrocytes treated with amphiphilic drugs.

<p>Erythrocytes were suspended in FITC-labeled dextran and incubated with the indicated amphiphilic drugs to induce endovesiculation. Upon washing, the uptake of fluorescent label was quantified by flow cytometry. Representative histograms are shown. The concentrations were 1.5, 0.4 and 0.375 mM (blue) and 3.0, 0.6 and 0.75 mM (green) for primaquine, chlorpromazine and imipramine, respectively (red is a control incubation without drug).</p

Microscopic comparison of patient’s and control erythrocytes in drug-induced endovesiculation.

<p>Erythrocytes of a PKAN+ patient (B and D) and a control donor (A and C) were treated with 3 mM primaquine (A and B) or 0.8 mM chlorpromazine (C and D) in the presence of FITC-dextran to monitor the formation of endovesicles by confocal microscopy. Representative phase contrast (left panels), fluorescence (middle panels) and overlay (right panels) images are shown.</p

Correlations between drug-induced endovesiculation, LPA-induced PS exposure and calcium uptake.

<p>The data of ChAc patients (circles) and respective control samples (triangles) for % of cells with endovesicles upon incubation with primaquine, for % annexin V-positive cells and % Fluo-3-positive cells upon LPA treatment are blotted against each other as indicated (data derived from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076715#pone-0076715-g004" target="_blank">Figures 4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076715#pone-0076715-g007" target="_blank">7</a>). Linear regression lines are shown with R2 (shown as inserts). In each combination a positive correlation is observed with Pearson’s r and the 2-tailed significance (given as inserts).</p

Differences in LPA-induced PS exposure and calcium uptake in erythrocytes of neuroacanthocytosis patients.

<p>Erythrocytes from ChAc, PKAN+ and PKAN- patients and control donors were treated with LPA as described in Materials and Methods and stained for PS exposure with FITC-annexin V (upper panels) or calcium uptake with Fluo-3 (lower panels) and analysed by flow cytometry. Overlays of representative histograms of patients (red) and controls (blue) show reduced PS exposure and calcium uptake in both ChAc and PKAN+ samples. The PKAN- sample does not differ from the respective control.</p

Altered LPA-induced PS exposure and calcium uptake in erythrocytes of patients with acanthocytes.

<p>Erythrocytes from patients and control donors were treated with LPA as described in Materials and Methods and either PS exposure (A) or calcium uptake (B) was monitored by flow cytometry. The percentage of FITC-annexin V-positive cells (A) and of Fluo-3-positive cells (B) of patient and control samples are shown and respective pairs are connected by lines. The data of the MPAN patient within the NBIA/PKAN- cohort is shown as a dashed line.</p