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Low-density lipoprotein mimics blood plasma-derived exosomes and microvesicles during isolation and detection.

Sódar BW, Kittel Á, Pálóczi K, Vukman KV, Osteikoetxea X, Szabó-Taylor K, Németh A, Sperlágh B, Baranyai T, Giricz Z, Wiener Z, Turiák L, Drahos L, Pállinger É, Vékey K, Ferdinandy P, Falus A, Buzás EI - Sci Rep (2016)

Bottom Line: Here we studied human pre-prandial and 4 hours postprandial platelet-free blood plasma samples as well as human platelet concentrates.Based on biophysical properties of LDL this finding was highly unexpected.Current state-of-the-art extracellular vesicle isolation and purification methods did not result in lipoprotein-free vesicle preparations from blood plasma or from platelet concentrates.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, 1085, Hungary.

ABSTRACT
Circulating extracellular vesicles have emerged as potential new biomarkers in a wide variety of diseases. Despite the increasing interest, their isolation and purification from body fluids remains challenging. Here we studied human pre-prandial and 4 hours postprandial platelet-free blood plasma samples as well as human platelet concentrates. Using flow cytometry, we found that the majority of circulating particles within the size range of extracellular vesicles lacked common vesicular markers. We identified most of these particles as lipoproteins (predominantly low-density lipoprotein, LDL) which mimicked the characteristics of extracellular vesicles and also co-purified with them. Based on biophysical properties of LDL this finding was highly unexpected. Current state-of-the-art extracellular vesicle isolation and purification methods did not result in lipoprotein-free vesicle preparations from blood plasma or from platelet concentrates. Furthermore, transmission electron microscopy showed an association of LDL with isolated vesicles upon in vitro mixing. This is the first study to show co-purification and in vitro association of LDL with extracellular vesicles and its interference with vesicle analysis. Our data point to the importance of careful study design and data interpretation in studies using blood-derived extracellular vesicles with special focus on potentially co-purified LDL.

No MeSH data available.


Related in: MedlinePlus

LDL mimics EVs during TRPS and FCM.(A) Commercially available human LDL analyzed by TRPS. Note that particles with different sizes were detected within the MV size range (100–800 nm) (measured on NP150 and NP300 nanopore membranes, black and gray bars, respectively). (B) FCM analysis of LDL particles conjugated to latex beads. Commercial LDL stained for apoB and apoCII, but not for the EV markers CD9 and CD63 (black filled histograms: antibody control, empty histograms: LDL). (C) LDL detection by FCM without bead conjugation at different dilutions. Note that the measured event number increases with the dilution, suggesting a swarm effect. (D) The signal obtained from commercial LDL is also partially sensitive to 0.1% Tx-100 at a physiological concentration (2 mg/mL) and in the 10 × diluted sample (0.2 mg/mL) as well (*P < 0.05, **P < 0.01, Mann-Whitney test).
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f6: LDL mimics EVs during TRPS and FCM.(A) Commercially available human LDL analyzed by TRPS. Note that particles with different sizes were detected within the MV size range (100–800 nm) (measured on NP150 and NP300 nanopore membranes, black and gray bars, respectively). (B) FCM analysis of LDL particles conjugated to latex beads. Commercial LDL stained for apoB and apoCII, but not for the EV markers CD9 and CD63 (black filled histograms: antibody control, empty histograms: LDL). (C) LDL detection by FCM without bead conjugation at different dilutions. Note that the measured event number increases with the dilution, suggesting a swarm effect. (D) The signal obtained from commercial LDL is also partially sensitive to 0.1% Tx-100 at a physiological concentration (2 mg/mL) and in the 10 × diluted sample (0.2 mg/mL) as well (*P < 0.05, **P < 0.01, Mann-Whitney test).

Mentions: To test the hypothesis that the apoB100 positive particles represent LDL, purified human LDL from a commercial source (both from Sigma-Aldrich and Merck) was assessed by TRPS as shown in Fig. 5a. Strikingly, the sizes of particles ranged from 100 to 500 nm possibly suggesting aggregation of the LDL particles (Fig. 6a). TRPS analysis of LDL at 2 mg/mL concentration (similar to that of blood plasma2526) resulted in a two orders of magnitude difference in the detected particle number between NP100 and NP200 (Supplementary Fig. S7). The concentration at the average diameter of 110 nm was 1.0E13/mL, while the estimated particle number of LDL in human plasma is reported to be approximately 1.0E14/mL26, suggesting that we still only see the tip of the iceberg. We also analyzed LDL particles conjugated onto latex beads by FCM, and detected apoB and apoCII positivity on the bead surface (Fig. 6b). As expected, in the case of LDL no signal was detected for the EV markers CD9 and CD63 (Fig. 6b). We next asked whether LDL particles were also detectable by FCM without bead conjugation, and surprisingly, LDL at a concentration of 2 mg/mL2526 was readily detected by FCM. We demonstrated that the signal was caused by a swarm effect of LDL particles. Using serial dilution of commercial LDL we have shown that the detected event number increased in the diluted LDL sample (Fig. 6c,d). A similar swarm effect has been reported to influence the FCM detection of EVs27. Indeed, we also demonstrated this swarm effect by testing a PFP sample (Supplementary Fig. S8). Furthermore, we showed that LDL at the above physiological concentration shared side-scattering properties with MVs isolated from human blood plasma both in fasting and in postprandial states (Supplementary Fig. S9). We also analyzed the detergent sensitivity of commercial LDL and found that LDL was partially sensitive to 0.1% Tx-100, possibly due to the disruption of lipoprotein aggregates by the detergent (Fig. 6d).


Low-density lipoprotein mimics blood plasma-derived exosomes and microvesicles during isolation and detection.

Sódar BW, Kittel Á, Pálóczi K, Vukman KV, Osteikoetxea X, Szabó-Taylor K, Németh A, Sperlágh B, Baranyai T, Giricz Z, Wiener Z, Turiák L, Drahos L, Pállinger É, Vékey K, Ferdinandy P, Falus A, Buzás EI - Sci Rep (2016)

LDL mimics EVs during TRPS and FCM.(A) Commercially available human LDL analyzed by TRPS. Note that particles with different sizes were detected within the MV size range (100–800 nm) (measured on NP150 and NP300 nanopore membranes, black and gray bars, respectively). (B) FCM analysis of LDL particles conjugated to latex beads. Commercial LDL stained for apoB and apoCII, but not for the EV markers CD9 and CD63 (black filled histograms: antibody control, empty histograms: LDL). (C) LDL detection by FCM without bead conjugation at different dilutions. Note that the measured event number increases with the dilution, suggesting a swarm effect. (D) The signal obtained from commercial LDL is also partially sensitive to 0.1% Tx-100 at a physiological concentration (2 mg/mL) and in the 10 × diluted sample (0.2 mg/mL) as well (*P < 0.05, **P < 0.01, Mann-Whitney test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834552&req=5

f6: LDL mimics EVs during TRPS and FCM.(A) Commercially available human LDL analyzed by TRPS. Note that particles with different sizes were detected within the MV size range (100–800 nm) (measured on NP150 and NP300 nanopore membranes, black and gray bars, respectively). (B) FCM analysis of LDL particles conjugated to latex beads. Commercial LDL stained for apoB and apoCII, but not for the EV markers CD9 and CD63 (black filled histograms: antibody control, empty histograms: LDL). (C) LDL detection by FCM without bead conjugation at different dilutions. Note that the measured event number increases with the dilution, suggesting a swarm effect. (D) The signal obtained from commercial LDL is also partially sensitive to 0.1% Tx-100 at a physiological concentration (2 mg/mL) and in the 10 × diluted sample (0.2 mg/mL) as well (*P < 0.05, **P < 0.01, Mann-Whitney test).
Mentions: To test the hypothesis that the apoB100 positive particles represent LDL, purified human LDL from a commercial source (both from Sigma-Aldrich and Merck) was assessed by TRPS as shown in Fig. 5a. Strikingly, the sizes of particles ranged from 100 to 500 nm possibly suggesting aggregation of the LDL particles (Fig. 6a). TRPS analysis of LDL at 2 mg/mL concentration (similar to that of blood plasma2526) resulted in a two orders of magnitude difference in the detected particle number between NP100 and NP200 (Supplementary Fig. S7). The concentration at the average diameter of 110 nm was 1.0E13/mL, while the estimated particle number of LDL in human plasma is reported to be approximately 1.0E14/mL26, suggesting that we still only see the tip of the iceberg. We also analyzed LDL particles conjugated onto latex beads by FCM, and detected apoB and apoCII positivity on the bead surface (Fig. 6b). As expected, in the case of LDL no signal was detected for the EV markers CD9 and CD63 (Fig. 6b). We next asked whether LDL particles were also detectable by FCM without bead conjugation, and surprisingly, LDL at a concentration of 2 mg/mL2526 was readily detected by FCM. We demonstrated that the signal was caused by a swarm effect of LDL particles. Using serial dilution of commercial LDL we have shown that the detected event number increased in the diluted LDL sample (Fig. 6c,d). A similar swarm effect has been reported to influence the FCM detection of EVs27. Indeed, we also demonstrated this swarm effect by testing a PFP sample (Supplementary Fig. S8). Furthermore, we showed that LDL at the above physiological concentration shared side-scattering properties with MVs isolated from human blood plasma both in fasting and in postprandial states (Supplementary Fig. S9). We also analyzed the detergent sensitivity of commercial LDL and found that LDL was partially sensitive to 0.1% Tx-100, possibly due to the disruption of lipoprotein aggregates by the detergent (Fig. 6d).

Bottom Line: Here we studied human pre-prandial and 4 hours postprandial platelet-free blood plasma samples as well as human platelet concentrates.Based on biophysical properties of LDL this finding was highly unexpected.Current state-of-the-art extracellular vesicle isolation and purification methods did not result in lipoprotein-free vesicle preparations from blood plasma or from platelet concentrates.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, 1085, Hungary.

ABSTRACT
Circulating extracellular vesicles have emerged as potential new biomarkers in a wide variety of diseases. Despite the increasing interest, their isolation and purification from body fluids remains challenging. Here we studied human pre-prandial and 4 hours postprandial platelet-free blood plasma samples as well as human platelet concentrates. Using flow cytometry, we found that the majority of circulating particles within the size range of extracellular vesicles lacked common vesicular markers. We identified most of these particles as lipoproteins (predominantly low-density lipoprotein, LDL) which mimicked the characteristics of extracellular vesicles and also co-purified with them. Based on biophysical properties of LDL this finding was highly unexpected. Current state-of-the-art extracellular vesicle isolation and purification methods did not result in lipoprotein-free vesicle preparations from blood plasma or from platelet concentrates. Furthermore, transmission electron microscopy showed an association of LDL with isolated vesicles upon in vitro mixing. This is the first study to show co-purification and in vitro association of LDL with extracellular vesicles and its interference with vesicle analysis. Our data point to the importance of careful study design and data interpretation in studies using blood-derived extracellular vesicles with special focus on potentially co-purified LDL.

No MeSH data available.


Related in: MedlinePlus