CoChat
Sign Up
Cited by 879MicroRNA in disease regulationExtracellular vesicles in diseaseCancer Genomics and Diagnostics

Blood Cell Origin of Circulating MicroRNAs: A Cautionary Note for Cancer Biomarker Studies

Open access
Pritchard, Colin C., Kroh, Evan, Wood, Brent, Arroyo, Jason D., Dougherty, Katy J., Miyaji, Melanie M., Tait, Jonathan F., Tewari, Muneesh·2012·Cancer Prevention Research
DOI 10.1158/1940-6207.capr-11-0370PMC PMC4186243

Published version of a preprint

Open this paper in CoChat — free account

Abstract

Circulating, cell-free microRNAs (miRNAs) hold great promise as a new class of cancer biomarkers due to their surprisingly high stability in plasma, association with disease states, and ease of sensitive measurement. Yet little is known about the origin of circulating miRNAs in either healthy or sick people or what factors influence levels of circulating miRNA biomarkers. Of 79 solid tumor circulating miRNA biomarkers reported in the literature, we found that 58% (46 of 79) are highly expressed in one or more blood cell type. Plasma levels of miRNA biomarkers expressed by myeloid (e.g., miR-223, miR-197, miR-574-3p, and let-7a) and lymphoid (e.g., miR-150) blood cells tightly correlated with corresponding white blood cell counts. Plasma miRNA biomarkers expressed by red blood cells (e.g., miR-486-5p, miR-451, miR-92a, and miR-16) could not be correlated to red cell counts due to limited variation in hematocrit in the cohort studied but were significantly increased in hemolyzed specimens (20- to 30-fold plasma increase; P < 0.0000001). Finally, in a patient undergoing autologous hematopoietic cell transplantation, plasma levels of myeloid- and lymphoid-expressed miRNAs (miR-223 and miR-150, respectively) tracked closely with changes in corresponding blood counts. We present evidence that blood cells are a major contributor to circulating miRNA and that perturbations in blood cell counts and hemolysis can alter plasma miRNA biomarker levels by up to 50-fold. Given that a majority of reported circulating miRNA cancer biomarkers are highly expressed in blood cells, we suggest caution in interpretation of such results as they may reflect a blood cell-based phenomenon rather than a cancer-specific origin. Cancer Prev Res; 5(3); 492–7. ©2011 AACR.

AI snapshot

Written from the paper’s full text.

TL;DR

Circulating microRNA biomarkers for solid tumors are frequently expressed by blood cells, and plasma levels of many such miRNAs track blood cell counts or hemolysis rather than tumor-specific biology. The authors caution that blood cell effects can be larger than reported cancer-vs-control differences and recommend interpreting circulating miRNA biomarkers alongside complete blood counts.

Key findings

  • Of 79 literature-reported solid tumor circulating miRNA biomarkers, 58% were highly expressed in one or more blood cell type; the abstract reports 47/79, while the Results section reports 46/79.
  • Plasma levels of myeloid-expressed miRNAs (e.g., miR-223, miR-197, miR-574-3p, let-7a) and lymphoid-expressed miR-150 correlated with corresponding white blood cell counts, with >50-fold differences in plasma miRNA levels between patients with the highest and lowest overall cell counts.
  • In hemolyzed specimens, all four RBC-associated miRNAs (miR-486-5p, miR-451, miR-92a, miR-16) increased 20–30 fold in plasma (p<0.0000001), whereas none of six non-RBC-associated miRNAs were significantly increased.
  • In a patient undergoing myeloablative chemotherapy and autologous hematopoietic stem cell transplant, plasma miR-223 and miR-150 tracked closely with changes in myeloid and lymphoid blood counts, respectively.

Methods

  • qRT-PCR profiling of 369 miRNAs was performed on FACS-sorted blood cell populations, plasma, and whole blood; 4 independent FACS experiments used peripheral blood from one healthy male donor.
  • A cohort of 42 consecutive residual patient plasma specimens with complete differential blood counts was used to correlate ten selected miRNAs with blood cell counts; one additional transplant patient had serial plasma samples collected prospectively.
  • Main analyses included correlation of plasma miRNA levels with blood cell counts, comparison of hemolyzed vs. non-hemolyzed specimens, and unsupervised hierarchical clustering of plasma miRNA expression across patient samples.

Limitations

  • Stated: the study does not directly assess the relative contribution of different blood cell types to plasma miRNA or examine mechanisms of miRNA release into plasma.
  • Stated: pre-analytical and analytical sources of variation, including collection, processing, RNA extraction, measurement, data acquisition, and normalization, may influence plasma miRNA levels.
  • Evident: RBC-expressed miRNAs could not be correlated with RBC counts because of limited variation in RBC count/hematocrit in the cohort; only 3 of 42 plasma specimens were hemolyzed; the transplant time-course involved a single patient; and the text contains minor numerical discrepancies (e.g., 47/79 vs. 46/79; 21 specimens vs. 25 timepoints; 32- vs. 33-day period).

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1158/1940-6207.capr-11-0370

Cites

34 of 34 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 7 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Plasma levels of the myeloid-expressed miRNAs let-7a, miR-223, miR-197, and miR-574-3p show significant positive correlations with myeloid blood cell counts (granulocytes and platelets).

    “Plasma levels of the myeloid-expressed miRNAs let-7a, miR-223, miR-197, and miR-574-3p showed significant positive correlations with myeloid blood cell counts (granulocytes and platelets) (Fig. 2B – left)”
    Quote in the paperSubstantial claim
  • Findingown data

    Three of the 42 plasma specimens were noted to be hemolyzed.

    “3 of the 42 plasma specimens were noted to be hemolyzed.”
    Quote in the paperSubstantial claim
  • Findingown data

    All four RBC-associated miRNAs were increased by 20-30 fold in hemolyzed plasma compared to non-hemolyzed specimens (p<0.0000001 in the Abstract).

    “all four RBC-associated miRNAs were increased by 20-30 fold in hemolyzed plasma”
    • Also stated in: § Abstract · plain
      “were significantly increased in hemolyzed specimens (20-30 fold plasma increase; p<0.0000001)”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Hemolysis determined by visual inspection accurately identifies specimens with red blood cell lysis sufficient to release miRNAs.
  • Findingown data

    The RBC-expressed miRNAs selected (miR-451, miR-92a, miR-16, and miR-486-5p) did not show correlations with white blood cell or platelet counts.

    “RBC-expressed miRNAs we had selected did not show correlations with white blood cell or platelet counts (Fig. 2B - left).”
    Quote in the paperSubstantial claim
  • Findingown data

    The distribution of miRNA expression values in plasma mirrors that of blood cells.

    “Comparing expression profiles of miRNAs detectable in plasma to miRNA profiles of blood cells demonstrated that the distribution of miRNA expression values in plasma mirrors that of blood cells (Fig. 1).”
    Quote in the paperSubstantial claim
  • May combine two claims. 5
  • Classified as probably not a substantial claim. 8

  • May combine two claims.

    Findingown data

    Plasma levels of liver-restricted miR-122 were not significantly correlated to blood cell counts or influenced by red blood cell hemolysis.

    “plasma levels of liver-restricted miR-122 that we selected as a negative control were not significantly correlated to blood cell counts or influenced by red blood cell hemolysis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Plasma levels of RBC-expressed miRNAs were tightly inter-correlated even among non-hemolyzed specimens, but not substantially correlated to six non-RBC expressed miRNAs examined.

    “plasma levels of RBC-expressed miRNAs were tightly inter-correlated even among non-hemolyzed specimens, but not substantially correlated to six non-RBC expressed miRNAs we examined (Figs. S2B and S2C).”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisenone given

    At least 79 miRNAs have been reported as plasma or serum miRNA biomarkers of solid tumors.

    “At least 79 miRNAs have been reported as plasma or serum miRNA biomarkers of solid tumors (i.e., non-hematopoietic malignancies), including prostate, lung, breast, colon, ovarian, esophageal, melanoma, and gastric cancer (Table S1).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    A total of 4 independent FACS experiments were performed on separate days using peripheral blood from the same healthy male volunteer donor.

    “A total of 4 independent FACS experiments were performed on a Becton Dickinson ARIA cell sorter on separate days, using peripheral blood from the same healthy male volunteer donor (H1-1).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    An additional 12 control arrays were performed on serial 2-fold dilutions of 30 μl of plasma from a single healthy donor to determine the analytic measurement ranges for each qPCR assay on the Exiqon array.

    “An additional 12 control arrays were performed on serial 2-fold dilutions of 30 μl of plasma from a single healthy donor to determine the analytic measurement ranges for each qPCR assay on the Exiqon array as previously described (7).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [7] Arroyo, 2011, Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma, Proc Natl Acad Sci U S A source

      Queued — the background check reaches it, usually within a day.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All plasma processing was performed in the specimen processing area of a large clinical laboratory using the same standardized plasma processing procedure applied to clinical specimens.

    “All plasma processing was performed in the specimen processing area of a large clinical laboratory using the same standardized plasma processing procedure applied to clinical specimens.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductnone given

    This study does not directly assess the relative contribution of different blood cell types to plasma miRNA or examine mechanisms of miRNA release into plasma.

    “This study does not directly assess the relative contribution of different blood cell types to plasma miRNA, or examine mechanisms of miRNA release into plasma.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    All plasma RNA preparations were made from 200μL in duplicate to control for variation in the RNA preparation step.

    “All plasma RNA preparations were made from 200μL in duplicate to control for variation in the RNA preparation step.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Blood cell abundance can influence circulating miRNA levels, as shown by in vivo evidence from myeloablative chemotherapy and hematopoietic stem cell transplant engraftment.

    “our studies in the setting of myeloablative chemotherapy and hematopoietic stem cell transplant engraftment provide the first direct, in vivo evidence that blood cell abundance can influence circulating miRNA levels.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Changes in plasma miRNA levels in the single transplant patient are attributable to changes in blood cell counts rather than other effects of myeloablative chemotherapy or transplant.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Elevated neutrophil counts are associated with shortened progression-free and overall survival in several cancers including non-small cell lung cancer and breast cancer.

    “elevated neutrophil counts are associated with shortened progression-free and overall survival in several cancers including non-small cell lung cancer and breast cancer (21, 22).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [21] Azab, 2011, Usefulness of the neutrophil-to-lymphocyte ratio in predicting short- and long-term mortality in breast cancer patients, Ann Surg Oncol source
    • cites [22] Teramukai, 2009, Pretreatment neutrophil count as an independent prognostic factor in advanced non-small-cell lung cancer: an analysis of Japan Multinational Trial Organisation LC00–03, Eur J Cancer source

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductnone given

    All plasma was processed fresh on the same day of blood draw and never frozen.

    “All plasma was processed fresh on the same day of blood draw (never frozen).”
    Quote in the paperSubstantial claim
11 of 11

  • The paper relies on this without support.

    Changes in plasma miRNA levels in the single transplant patient are attributable to changes in blood cell counts rather than other effects of myeloablative chemotherapy or transplant.

    “we examined miR-223 (myeloid cell-expressed) and miR-150 (lymphoid cell-expressed) miRNAs in the plasma of a patient over a time-course of myeloablative chemotherapy and hematopoietic stem cell transplant engraftment (Table S3).”
    “demonstrating that white blood cell counts can significantly influence plasma levels of blood cell-expressed miRNAs.”

  • The paper relies on this without support.

    Blood cell miRNA expression profiles measured in purified sorted blood cells reflect the contribution of those cell types to circulating plasma miRNAs in vivo.

    “We used qRT-PCR to examine expression of 79 miRNAs reported as circulating solid tumor biomarkers in purified sub-populations of blood cells and in matched healthy donor plasma (9-13) (Fig. S1).”
    “suggesting that blood cells could be a major source for these plasma miRNAs”

  • The paper relies on this without support.

    The 42 residual patient specimens are representative of a general patient population for assessing correlations between blood cell counts and plasma miRNA levels.

    “The cohort consisted of consecutive residual patient plasma samples collected at a single academic medical center, including both inpatients and outpatients with a wide variety of underlying diseases (Table S2).”

  • The paper relies on this without support.

    Hemolysis determined by visual inspection accurately identifies specimens with red blood cell lysis sufficient to release miRNAs.

    “specimen characteristics (such as hemolysis determined by visual inspection)”
    “3 of the 42 plasma specimens were noted to be hemolyzed.”

  • The paper relies on this without support.

    The 79 literature-reported solid tumor biomarkers are a representative set of circulating miRNA cancer biomarkers.

    “At least 79 miRNAs have been reported as plasma or serum miRNA biomarkers of solid tumors (i.e., non-hematopoietic malignancies), including prostate, lung, breast, colon, ovarian, esophageal, melanoma, and gastric cancer (Table S1).”
5 of 5

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Most plasma miRNAs were RBC derived, followed by platelets, mesenchymal cells, and immune cells.

    “Similar to a recent manuscript on cell-free RNA in plasma [40] and consistent with other miRNA-based studies [41–43], we observed that most plasma miRNAs were RBC derived, followed by platelets, mesenchymal cells, and immune cells.”

    From A curated human cellular microRNAome based on 196 primary cell types (2022) · cites this paper as [43]

    The Results report that blood cells could be a major source of plasma miRNAs and that RBC-associated miRNAs increase substantially in hemolyzed plasma. But the Discussion explicitly says the study did not assess the relative contributions of different blood cell types. It therefore does not support RBC predominance or the stated ranking of platelets, mesenchymal cells, and immune cells. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • Many erythrocyte-enriched miRNAs also appear to be abundant in epithelia.

    “However, many such miRNAs also appear to be abundant in epithelia (Supplemental Data 1) [22]”

    From Novel Approach to Fecal Occult Blood Testing by Assay of Erythrocyte-Specific microRNA Markers (2017) · cites this paper as [22]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Reported biomarker percentage does not match its fraction

    Major

    The abstract reports 58% as 47/79, but 47/79 is approximately 59.5%. The Results section instead reports 46/79, which is approximately 58%.

    Reported 58 · recomputed 59.493671

  • Serial specimen and timepoint counts disagree

    Minor

    The methods state that 21 residual plasma specimens were collected from the transplant patient, while the Results state that serial plasma samples and blood counts were collected at 25 timepoints for that patient.

2 of 2

Cited works that were retracted

Works this paper cites that were later retracted, from the retraction notices publishers deposit. Major when the notice came out before this paper was published.

  • Cites a retracted work: 10.1016/j.ygyno.2008.04.033

    Info

    The cited work was retracted on 2023-07-20 (notice 10.1016/j.ygyno.2023.06.002), not before this paper was published (2012-03-01).

    Cited work 10.1016/j.ygyno.2008.04.033 · retracted 2023-07-20 · notice 10.1016/j.ygyno.2023.06.002 · this paper published 2012-03-01

1 of 1

Cites

1 retracted

Flagged first, then by citations

Research works printed but not linked: 1

  1. Heegaard, 2011, Circulating microRNA expression profiles in early stage non-small cell lung cancer, Int J Cancer

Cited by

Sorted by citations

Related

Authors
Pritchard, C. C., Kroh, E., Wood, B., Arroyo, J. D., Dougherty, K. J., Miyaji, M. M., Tait, J. F., & Tewari, M.
Affiliations
University of Washington (US)
Corresponding author
Tewari, Muneesh
ORCID
Venue
Cancer Prevention Research
Publisher
American Association for Cancer Research (AACR)
Published
2012-03-01
Volume
5
Issue
3
Pages
492-497
ISSN
  • 1940-6207 (ISSN-L)
  • 1940-6215
DOI
10.1158/1940-6207.capr-11-0370
PMC
PMC4186243
OpenAlex
W2140775205
Cited by
879
References
34
Citations by year
2026: 29 · 2025: 28 · 2024: 36 · 2023: 25 · 2022: 45 · 2021: 61 · 2020: 59 · 2019: 56 · 2018: 71 · 2017: 86 · 2016: 87 · 2015: 103 · 2014: 95 · 2013: 71 · 2012: 27
FWCI
20.63
Type
journal article
Language
en
Open access
Locations
  • Cancer Prevention Research (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • National Institutes of Health (5p50ca083636-15)
  • National Cancer Institute (P50-CA083636, P30CA015704, P50CA097186)
  • National Institute of Diabetes and Digestive and Kidney Diseases (R01DK085714)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer Genomics and Diagnostics
Keywords
circulating microRNAs, cell-free microRNAs, tumor markers, hemolysis, white blood cell count, red blood cell count, myeloid cells, lymphoid cells, hematopoietic stem cell transplantation, miR-223, miR-150, miR-574-3p, miR-197, miR-451, miR-486-5p, miR-92a, miR-16
MeSH terms
Erythrocytes, Humans, Neoplasms, Plasma, RNA, Messenger, Biomarkers, Tumor, Oligonucleotide Array Sequence Analysis, Gene Expression Profiling, MicroRNAs, Real-Time Polymerase Chain Reaction

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-11-06.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-11-06.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review