Article Figures & Data
Figures
- Fig. 1. Liposome capture by the filter plate.
Fluorescent liposomes (0–1.13 μg) were suspended in 100 μL PBS, pH 7.4, and applied to the filter plate and centrifuged at 2000g for 5 min. Fluorescent intensities of the liposome were measured before (□) and after (
) filtration, and capture efficiencies were calculated (○). Inset, SEM images of liposomes. - Fig. 2. Method validation.
(A, B), SEM analysis. After pooled human plasma was applied to the filter plate, filter membranes were removed, air dried, sputter coated with gold, and imaged at an accelerating voltage of 5 kV (A) and 3 kV (B) using the lower and upper secondary electron detectors, respectively [top surface (A), cross section (B)]. Arrows indicate EMV-like particles with a diameter of approximately 100 nm. (C), Dose responses. Plasma (10–1000 μL) was applied to the filter plate, and various mRNAs were measured by our method. Each symbol was the mean of the Ct values from triplicate plasma samples. ○, ACTB; Δ, B2M; ■, ITGA2B; ◊, UROD; •, HBB; ▴, SRGN; ♦, DEFA3; – · –, theoretical slope. (D), Comparison between ultracentrifugation and our method. Plasma (300 μL) was ultracentrifuged at 100 000g for 2 h, then the pellets were resuspended in our lysis buffer, followed by mRNA analysis by our method. The results (y axis) were compared with the data of our filter plate method (x axis) using the identical plasma samples. Graphical symbols are the same as in Fig. 2C. (E), Reproducibility. Using the same plasma samples (300 μL, triplicate), our method was repeated 3 times. Graphical symbols are the same as in Fig. 2C. The bars indicate SD. Exp., experiment.
- Fig. 3. Comparison between EMV mRNA analysis and conventional CBC, typical case.
The upper panels (A–C) plot the levels of EMV mRNAs [(A), DEFA3 (○) and SRGN (•); (B), HBB (○) and UROD (•); (C), ITGA2B (○) and ITGB3 (•)]. The bottom panels (D–F) plot the results of CBC analysis from a single typical case [(D), WBC; (E), reticulocytes; (F), platelets]. In (A–C), the results of PCR (Ct) were converted to the copy number per milliliter plasma as described in the Materials and Methods. Vertical dashed lines indicate day 7, when neither mRNA or CBC results could detect BM recovery, whereas the arrows indicate the points where mRNA was first recovered.
- Fig. 4. Comparison between EMV mRNA analysis and conventional assays.
(A–C), EMV mRNA vs CBC for all 18 cases combined. The recovery days of EMV mRNA (first detectable mRNA) are shown on the x axis, and CBC recovery days (WBC, >500/μL; reticulocytes, >4/μL; platelets, >20 000 without blood transfusion) are shown on the y axis, up to day 30. (A), WBC vs DEFA3; (B), RBC vs HBB; (C), platelets vs ITGA2B. Dotted lines are plotted at an angle of 45°. The data points above this line represent the patients who demonstrated earlier mRNA evidence of recovery than CBC. Small dots represent a single patient, and large dots represent multiple patients at the same location. The number of patients for each large dot is shown by arrows. (D–F), EMV mRNA analysis vs BM data at day 14–15. The recovery days of EMV mRNA (x axis) were the same as those shown in (A–C). The y axis represents nucleated cell counts (NCC) in BM aspirations at day 14–15 (n = 11). (A), NCC vs DEFA3; (B), NCC vs HBB; (C), NCC vs ITGA2B. Solid lines indicate regression lines with r2 values. Small and large dots are the same as (A–C).
Tables
Case no. Age, years Sex Disease Initial disease status Donor Conditioning CMV reactivation, yes/no Acute GvHD, grade 1 33 M Ph+ALLa CR2 RPB M No No 2 38 M MDS RCMD BM M Yes No 3 16 M SAA TD BM M Yes 2 4 71 F AML CR3 BM Non-M Yes 1 5 60 M MDS RCMD BM M Yes 2 6 25 M ALL CR1 BM M Yes 2 7 39 F AML CR1 RPB M No 1 8 39 F Ph+ALL CR1 CB M Yes 1 9 28 M SAA TD BM M Yes 2 10 33 F AML CR2 post BMT BM M No No 11 37 M AML RL1 post BMT BM M Yes 1 12 60 M AML CR1 BM M Yes 1 13 28 M Ph+ALL CR1 CB M No 1 14 38 M ALL RL1 BM M No 1 15 60 M MDS RAEB 1 RBM M Yes 2 16 33 F AML RL2 BM M No 2 17 62 M ALL CR1 BM M Yes No 18 46 F AML RL1 CB M No 1 ↵a Ph+ALL, Philadelphia chromosome-positive acute lymphoblastic leukemia; MDS, myelodysplastic syndrome; SAA, severe aplastic anemia; AML, acute myeloid leukemia; CR2, second CR; RCMD, refractory cytopenia with multilineage dysplasia; TD, transfusion dependent; RL1, first relapse; BMT, BM transplantation; RAEB, refractory anemia with excessive blast; RPB, related PB; CB, cord blood; M, myeloablative; CMV, cytomegalovirus; GvHD, graft vs host disease.
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