Table 1.
Calculated median cell counts and viabilities of PBMCs isolated from blood samples.
Fig 1.
PBMC analysis in matched pairs collected in different anticoagulants.
104 randomly selected, matched blood samples were Heparin- and EDTA-anticoagulated followed by PBMC isolation. A) shows scatterplot of cell numbers and B) displays a Bland-Altman plot indicating the percentage bias between the two coagulants.
Fig 2.
Frequency of defined immune cells in isolated PBMCs assessed by epigenetic qPCR.
Lymphocyte frequencies (left Y-scale) and numbers (right Y-scale) are depicted in red and result from adding T, B and NK cell counts. Granulocytes (blue) and monocytes (green) are also assessed over time. Overall, the absolute cell count is shown in grey. All analyses were performed with PBMCs isolated from (A) EDTA- and (B) Heparin- anticoagulated blood. Medians for each cell type are displayed at each time point. Per cell type medians were connected to display trend over time.
Fig 3.
Lymphocyte analysis by Flow cytometry and epigenetic qPCR on aging PBMC preparations.
PBMC samples from blood drawn into EDTA and Heparin are shown in the upper and lower row, respectively. In the left column, lymphocyte counts as assessed by flow (y-axis) and epigenetics (x-axis). In the middle column, the respective Bland-Altman plot, displaying relative differences (y-axis) over average of each measurement (x-axis), is shown. Samples purified before and after the changepoint are indicated in black and green, respectively. On the right-hand side, Box-Plots of the percent difference for all data assessed prior to and after the changepoint, determined by PELT are given. Box-Plots according to Tukey, outlier not shown.
Fig 4.
Granulocyte analysis by Flow cytometry and epigenetic qPCR in aging PBMC preparations.
PBMC samples from blood drawn into EDTA and Heparin are shown in the upper and lower row, respectively. In the left column, granulocytes were assessed by flow (y-axis) and epigenetics (x-axis). In the middle column, the according Bland-Altman plots display relative differences (y-axis) over average of each measurement (x-axis). Samples purified before and after the changepoint are indicated in black and green, respectively. The very low granulocyte content in early vs the relatively high counts after prolonged incubation are a challenge for the method comparison. On the right-hand side, boxplots of the percentage difference for all data assessed prior to and after the PELT changepoint are given. Box-Plots according to Tukey, outlier not shown.
Fig 5.
Monocyte analysis by Flow cytometry and epigenetic qPCR on aging PBMC preparations.
PBMC samples from blood drawn into EDTA and Heparin are shown in the upper and lower row, respectively. In the left column, monocytes were assessed by flow cytometry (y-axis) and epigenetic qPCR (x-axis). In the middle column, the according Bland-Altman plots display percentage differences (y-axis) over average of each measurement (x-axis). Samples purified before and after the changepoint are indicated in black and green, respectively. On the right-hand side, Box-Plots of the percent differences for all data assessed prior to and after the PELT changepoint are given. Box-Plots according to Tukey, outlier not shown.
Fig 6.
Granulocyte presence in PBMC isolates measured by epigenetic qPCR.
SepMate was used to purify PBMCs after initial storage of Heparin anticoagulated whole blood from four donors at 4°C, room temperature and 37°C for 12, 24 or 48 hours. All four data points are shown, and means are indicated; whisker added to visually separate category.
Fig 7.
PBMC quality dependence on isolation protocol and matrix.
Standard performance indicators, cell count (recovery) and viability, were tested. For a matched sample cohort with 9 donors, PBMCs were isolated 0 and 72 hours after blood draw using the Sepmate protocol (SepM) or the Ficoll overlay method (CTL). On a second, non-matched cohort, PBMCs from 48 donors were prepared within 12 hours of blood draw. Boxes are given by the range of the third (Q3) to the first quartile (Q1). The median is displayed in the middle of the box. The whiskers extend to the max (min) observation or 1.5 times interquartile range above Q3 (below Q1). Observations displayed as jittered scatter in their respective category.
Table 2.
Epigenetic immune cell counting in PBMCs isolated from aged blood.