TY - JOUR
T1 - Zoom in on antibody aggregates
T2 - A potential pitfall in the search of rare EV populations
AU - Rasmussen, Rikke W.
AU - Botha, Jaco
AU - Prip, Frederik
AU - Sanden, Mathilde
AU - Nielsen, Morten H.
AU - Handberg, Aase
PY - 2021/2
Y1 - 2021/2
N2 - High‐resolution flow cytometers (hFCM) are used for the detection of extracellular vesicles (EV) in various biological fluids. Due to the increased sensitivity of hFCM, new artifacts with the potential of interfering with data interpretation are introduced, such as detection of antibody aggregates. The aim of this study was to investigate the extent of aggregates in labels commonly used for the characterization of EVs by hFCM. Furthermore, we aimed to compare the efficacy of centrifugation and filtering treatments to remove aggregates, as well as to quantify the effect of the treatments in reducing aggregates. For this purpose, we labeled phosphate buffered saline (PBS) with fluorescently conjugated protein labels and antibodies after submitting them to 5, 10, or 30 min centrifugation, filtering or washed filtering. We investigated samples by hFCM and quantified the amount of aggregates found in PBS labeled with untreated and pre‐treated labels. We found a var-ying amount of aggregates in all labels investigated, and further that filtering is most efficient in removing all but the smallest aggregates. Filtering protein labels can reduce the extent of aggregates; however, how much remains depends on the specific labels and their combination. Therefore, it is still necessary to include appropriate controls in a hFCM study of EVs.
AB - High‐resolution flow cytometers (hFCM) are used for the detection of extracellular vesicles (EV) in various biological fluids. Due to the increased sensitivity of hFCM, new artifacts with the potential of interfering with data interpretation are introduced, such as detection of antibody aggregates. The aim of this study was to investigate the extent of aggregates in labels commonly used for the characterization of EVs by hFCM. Furthermore, we aimed to compare the efficacy of centrifugation and filtering treatments to remove aggregates, as well as to quantify the effect of the treatments in reducing aggregates. For this purpose, we labeled phosphate buffered saline (PBS) with fluorescently conjugated protein labels and antibodies after submitting them to 5, 10, or 30 min centrifugation, filtering or washed filtering. We investigated samples by hFCM and quantified the amount of aggregates found in PBS labeled with untreated and pre‐treated labels. We found a var-ying amount of aggregates in all labels investigated, and further that filtering is most efficient in removing all but the smallest aggregates. Filtering protein labels can reduce the extent of aggregates; however, how much remains depends on the specific labels and their combination. Therefore, it is still necessary to include appropriate controls in a hFCM study of EVs.
KW - Extracellular vesicles
KW - High‐resolution flow cytometry
KW - Protein aggregates
UR - http://www.scopus.com/inward/record.url?scp=85102100464&partnerID=8YFLogxK
U2 - 10.3390/biomedicines9020206
DO - 10.3390/biomedicines9020206
M3 - Journal article
AN - SCOPUS:85102100464
SN - 2227-9059
VL - 9
JO - Biomedicines
JF - Biomedicines
IS - 2
M1 - 206
ER -