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Research ArticleArticle

Posttransplantation Bone Marrow Assessment by Quantifying Hematopoietic Cell–Derived mRNAs in Plasma Exosomes/Microvesicles

Jun Aoki, Kazuteru Ohashi, Masato Mitsuhashi, Taku Murakami, Melanie Oakes, Takeshi Kobayashi, Noriko Doki, Kazuhiko Kakihana, Hisashi Sakamaki
DOI: 10.1373/clinchem.2013.213850 Published March 2014
Jun Aoki
Hematology Division, Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Tokyo, Japan;
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Kazuteru Ohashi
Hematology Division, Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Tokyo, Japan;
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Masato Mitsuhashi
Hitachi Chemical Research Center, Irvine, CA.
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  • For correspondence: mmitsuhashi@hcrcenter.com
Taku Murakami
Hitachi Chemical Research Center, Irvine, CA.
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Melanie Oakes
Hitachi Chemical Research Center, Irvine, CA.
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Takeshi Kobayashi
Hematology Division, Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Tokyo, Japan;
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Noriko Doki
Hematology Division, Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Tokyo, Japan;
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Kazuhiko Kakihana
Hematology Division, Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Tokyo, Japan;
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Hisashi Sakamaki
Hematology Division, Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Tokyo, Japan;
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    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 (Embedded Image) filtration, and capture efficiencies were calculated (○). Inset, SEM images of liposomes.

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    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.

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    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.

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    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).

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    Table 1. Patient characteristics.
    Case no.Age, yearsSexDiseaseInitial disease statusDonorConditioningCMV reactivation, yes/noAcute GvHD, grade
    133MPh+ALLaCR2RPBMNoNo
    238MMDSRCMDBMMYesNo
    316MSAATDBMMYes2
    471FAMLCR3BMNon-MYes1
    560MMDSRCMDBMMYes2
    625MALLCR1BMMYes2
    739FAMLCR1RPBMNo1
    839FPh+ALLCR1CBMYes1
    928MSAATDBMMYes2
    1033FAMLCR2 post BMTBMMNoNo
    1137MAMLRL1 post BMTBMMYes1
    1260MAMLCR1BMMYes1
    1328MPh+ALLCR1CBMNo1
    1438MALLRL1BMMNo1
    1560MMDSRAEB 1RBMMYes2
    1633FAMLRL2BMMNo2
    1762MALLCR1BMMYesNo
    1846FAMLRL1CBMNo1
    • ↵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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    Table 2. Correlations among EMV mRNA, CBC, and clinical outcomes.
    RNA elevation, daysaCBC recovery, nStatus on day 180, n
    ≤30 daysb>30 daysCR/NHRL/TDc
    WBC≤1511083
    >156143
    RBC≤156060
    >1521066
    Platelet≤156060
    >1511166
    • ↵a RNA elevation, DEFA3 for WBC, HBB for RBC, and ITGA2B for platelets, respectively.

    • ↵b CBC recovery was WBC >500/μL, reticulocyte >3×104/μL, and platelets >20,000/μL.

    • ↵c RL, relapse; TD, transfusion dependent.

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Clinical Chemistry: 60 (4)
Vol. 60, Issue 4
April 2014
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Posttransplantation Bone Marrow Assessment by Quantifying Hematopoietic Cell–Derived mRNAs in Plasma Exosomes/Microvesicles
Jun Aoki, Kazuteru Ohashi, Masato Mitsuhashi, Taku Murakami, Melanie Oakes, Takeshi Kobayashi, Noriko Doki, Kazuhiko Kakihana, Hisashi Sakamaki
Clinical Chemistry Apr 2014, 60 (4) 675-682; DOI: 10.1373/clinchem.2013.213850
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Posttransplantation Bone Marrow Assessment by Quantifying Hematopoietic Cell–Derived mRNAs in Plasma Exosomes/Microvesicles
Jun Aoki, Kazuteru Ohashi, Masato Mitsuhashi, Taku Murakami, Melanie Oakes, Takeshi Kobayashi, Noriko Doki, Kazuhiko Kakihana, Hisashi Sakamaki
Clinical Chemistry Apr 2014, 60 (4) 675-682; DOI: 10.1373/clinchem.2013.213850

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