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J Clin Apher. Author manuscript; available in PMC 2020 December 12.
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Published in final edited form as:

J Clin Apher. 2016 December ; 31(6): 551–558. doi:10.1002/jca.21448.

Evidence of Relative Iron Deficiency in Platelet- and Plasma-

Pheresis Donors Correlates With Donation Frequency
Huihui Li1, Frances Condon1, Debra Kessler1, Vijay Nandi1, Mark Rebosa2, Mark
Westerman3, Beth H. Shaz1, Yelena Ginzburg1,*
1Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York
2Blood Operations, New York Blood Center, New York, New York
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3Intrinsic Lifesciences, LLC, LaJolla, California

Abstract
Background: The loss of iron stores and resulting iron deficiency is well documented in whole
blood or red blood cell donors. We hypothesized that relative iron deficiency also occurs as a result
of more frequent platelet- and plasma-pheresis (apheresis) donation.

Materials and Methods: To test this hypothesis, we proposed a pilot cross-sectional study to
analyze erythropoiesis- and iron-related parameters in white male apheresis donors: (1) relative to
controls, (2) in correlation with apheresis donation frequency, and (3) in correlation with pre-
donation platelet count.
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Results: Fifty eligible apheresis donors and eight controls were enrolled in the study. Apheresis
donors were found to have a lower serum ferritin and serum hepcidin and exhibited evidence of
iron restricted erythropoiesis relative to controls. Furthermore, among donors, lower MCV, CHr,
hepcidin concentration, and serum ferritin were observed in more frequent apheresis donors.
Correlations between donation frequency and hepcidin and ferritin were noted in apheresis donors.

Conclusions: This pilot study demonstrates that apheresis donors are relatively iron deficient
compared to controls and supports the premise that frequent apheresis donation correlates with
relatively iron restricted erythropoiesis. An analysis of iron- and erythropoiesis-related parameters
in a broader population of frequent apheresis donors (i.e., female and non-white donors) may
demonstrate larger deficits and an even greater potential benefit of iron replacement.

Keywords
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platelet- and plasma-pheresis donors; relative iron deficiency and iron restricted erythropoiesis;
apheresis donation frequency

*
Correspondence to: Yelena Ginzburg, Erythropoiesis Laboratory, New York Blood Center, 310 East 67th Street, New York, NY
10065. yginzburg@nybloodcenter.org.
Additional Supporting Information may be found in the online version of this article.
 Li et al. Page 2

INTRODUCTION
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Iron is an essential nutrient for most living organisms, and iron deficiency (ID) is the cause
of half of all anemias worldwide. Although iron is required pre-dominantly for adequate
hemoglobin synthesis, ID may result in abnormal DNA synthesis, immune response,
oxidative metabolism, and mitochondrial electron transport in addition to anemia. ID in the
absence of anemia has been linked to decreased muscle function in experimental animals
[1,2], cognitive impairment and fatigue in non-anemic women [3,4], and decline in physical
performance, which are all reversible with oral iron supplementation [5–7].

In contrast with “absolute ID,” defined by serum ferritin ≤ 22 ng/mL, relative “iron
depletion” typically occurs when serum ferritin is 22–40 mg/dL [8,9], correlating well with
depleted bone marrow iron stores [10,11], and, if untreated, may progress to iron restricted
erythropoiesis (IRE), defined by elevated serum soluble transferrin receptor 1 (sTfR1), and
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ultimately, ID anemia [8,12]. Additional parameters such as log(sTfR1/ferritin) have been

described and used to define the proportionality between erythroid iron demand (sTfR1) and
iron stores (serum ferritin). Furthermore, hepcidin has recently been described as the main
negative iron regulatory hormone, decreasing as iron demand increases, enabling iron
absorption and recycling [13,14]. How these parameters change relative to apheresis
donation frequency is incompletely understood.

Frequent blood donation may result in ID, and studies find absent iron stores and IRE in
repeat whole blood donors [12]. Measurement of serum ferritin, iron replacement therapy,
and prolongation of the inter-donation interval have been proposed to evaluate, treat, and
prevent, respectively, ID in blood donors [15]. Much of this discussion is focused on whole
blood and red blood cell (RBC) donors. Less is known about platelet- and plasma-pheresis
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(apheresis) donors, in whom consideration of ID or iron depletion without anemia is also

warranted. ID in apheresis donors is plausible because most apheresis donors have
previously donated whole blood [16,17], and as ID is associated with thrombocytosis
[16,18,19], those successfully converted from whole blood donors may exhibit ID, iron
depletion, or IRE without anemia. Furthermore, tubes taken for donor testing and residua in
the tubing (approximately 30 mL RBC loss in total [16]) at each donation results in iron loss
equivalent to 4 whole blood units annually in frequent apheresis donors (up to every 72 h, 24
times per year).

In addition, the mechanistic association between ID and thrombocytosis is incompletely

understood, but may be related to an inhibitory effect of iron on megakaryocytes in humans
[9] possibly as a consequence changes in megakaryocyte mitotic rate or time to complete
maturation and platelet release [20]. Although normalization of platelet count is consistently
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reported following successful treatment with iron [21–23] in both humans and rats, prior
studies demonstrate no correlation between platelet count and serum ferritin [7,9,23]. We
hypothesize that, because ID is associated with thrombocytosis, apheresis donors with
relative ID or IRE will also have the highest platelet counts.

Last, several publications provide conflicting data regarding the correlation between
donation frequency and depleted iron stores [17,24]. We hypothesize that frequent apheresis

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donors exhibit ID or iron depletion and that donation frequency correlates with IRE. The
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main purpose of analyzing these additional parameters as harbingers of anemia is to identify

donors at risk for potential ID effects of frequent apheresis donation, not traditionally
associated with risk of ID. We thus propose a pilot study to use more novel iron- and
erythropoiesis-related parameters (e.g., sTfR1, log(sTfR1/ferritin), and serum hepcidin) to
extend our understanding of the prevalence and physiology of ID and IRE in apheresis
donors and identify at-risk donors in advance of anemia and low hemoglobin deferral.

MATERIALS AND METHODS

Study Participants
All study participants were recruited from the population of donors at our donor center.
Goals of donor recruitment for our pilot study were to enroll study participants with a broad
but typical distribution of donation frequencies, excluding those with a history of iron-
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related pathology (i.e., ID, hereditary hemochromatosis, anemia, bleeding, or abnormal

colonoscopy findings); all donors were asked whether they take multi-vitamins and/or iron
supplementation. Male donors were selected for convenience because of the greater
variability in female donors regarding menstrual/parity/menopausal status and the restriction
of apheresis donation based on TRALI mitigation strategy, the later complicating
recruitment by reducing the pool of potentially eligible female apheresis donors.

Eligible study donors were white males age 40–65 years who donated by pheresis without
whole blood/RBCs donations within 2 years of study enrollment. Those who had not
donated any blood products in the prior 2 years were enrolled as controls (i.e., ineligible due
to travel or other parameters unrelated to iron and erythropoiesis) when they presented for
donation. Sample size was selected based on our power calculation for assessing correlation
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between donation frequency and iron- and erythropoiesis-related parameters; based on our
assessment of Hb, platelet count, and donation frequencies of white male apheresis donors at
our donor center, there is a power of 0.8 to detect differences (P < 0.05) and correlations (r >
0.3) with n = 50. All participants signed IRB approved informed consent. Platelet- and
plasma-pheresis were performed using Trima Accel separators (CaridianBCT, Zug,
Switzerland) with an estimated RBC loss of 30 mL [16]. Donation frequency was
determined as number of donations within 12 months prior to study enrollment.

Assays
Circulating RBC parameters (e.g., RBC count, hemoglobin, hematocrit, MCV, MCH,
MCHC, RDW, platelet and reticulocyte count, and CHr) were analyzed using Advia 120
Hematology System (Bayer Diagnostics, Tarrytown, NY). Serum iron, transferrin,
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erythropoietin, and unbound iron binding capacity (UIBC) were measured using standard
clinical laboratory (Jacobi Medical Center, Bronx, NY). Total iron binding capacity (TIBC)
and transferrin saturation were calculated using standard formula (UIBC + serum iron) and
(serum iron/TIBC) x 100, respectively. Serum hepcidin (Intrinsic Lifesciences, LLC,
LaJolla, CA) [14] and soluble serum TfR1 (R&D Systems, Minneapolis, MN) analyses were
performed using ELISA. As previously published, we define absolute ID as serum ferritin <
22 ng/mL [8,12] and IRE as log(sTfR1/ferritin) ≥ 1.25, the latter corresponding to the 97.5%

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of log(sTfR1/ferritin) distribution in the study population [12,25]. Log(sTfR1/ferritin) was

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calculated as the base 10 logarithm of the ratio between sTfR1 (in μg/mL) to ferritin (in ng/
mL).

Statistical Analysis
We analyze correlations of erythropoiesis- and iron-related parameters with donation
frequency as well as platelet count. Using Microsoft Excel and SAS version 9.3, Pearson
correlation coefficients were generated to examine the strength and direction of the
relationships between measures. Generally, a correlation coefficient ≥ 0.5 is strong, 0.3 –
0.49 is intermediate strength, and < 0.3 is weak [26]. Student’s t test compared arithmetic
mean differences between groups and Fisher’s exact test compared proportions. χ2 test was
used to determine the difference between the expected frequencies and the observed
frequencies of different parameters in blood donors. Data are presented as mean ± standard
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error with P ≤ 0.05 considered statistically significant.

RESULTS
Study Donors
Fifty eligible study donors and eight controls were enrolled in the study. Seven study donors
and 3 controls were incorrectly assigned as eligible, resulting in 43 study donors and 5
controls in our analyses. We compared eligible and ineligible donors to avoid potential
confounding and found no differences in age, number of donations, number of deferrals,
erythropoiesis- and iron-related parameters (Supporting Information Tables I and II). No
statistically significant differences in population characteristics were observed between
study donors and controls (Table I).
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Study Controls
Controls were all lapsed blood donors who donated whole blood/RBCs 9 ± 2 (range, 4–13)
years relative to study donors who donated 4 ± 0.3 (range, 2–14) years prior to study
enrollment (P < 0.0001). We explored the validity of our control group in comparison to
NHANES database (2001–2002), selecting a comparable subset of white males age 40–65 to
analyze available parameters and identified 500 individuals with data for iron related
parameters. No differences in serum ferritin (230 ± 86 vs.196 ± 9 ng/mL, P = 0.7) or total
iron binding capacity (TIBC) (311 ± 21 vs.359 ± 4 μg/dL, P = 0.2) are evident between
samples from our study-specific controls and the NHANES population [27]. Serum ferritin
and TIBC are reliable measured parameters, independent of sampling relative to dietary iron
intake. Data regarding sTfR1 was available only for pregnant females and hepcidin data is
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not included in the NHANES dataset [27].

Evidence of Relative Iron Deficiency and Iron Restricted Erythropoiesis in Apheresis

Donors
We used novel methods not previously assessed in apheresis donors to understand the
prevalence of ID and IRE. Study donors exhibit lower serum ferritin, transferrin saturation,
serum iron, and serum hepcidin; no cases of serum ferritin < 12 mg/dL (data not shown) and
no difference in frequency with serum ferritin < 22 mg/dL or < 40 mg/dL occurs in donors

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relative to controls (Table II). Furthermore, study donors exhibit a decreased log(sTfR1/
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ferritin), suggesting relative IRE (Table III). Two study donors (who donated 8 and 15 times
within 12 months of study enrollment, respectively) demonstrated evidence of IRE (Fig. 1a).
With the exception of a higher RBC count, no differences were observed in any circulated
RBC- or reticulocyte-related parameters or serum erythropoietin between study donors and
controls (Table III).

Correlation of Relative Iron Deficiency and Iron Restricted Erythropoiesis With Donation
Frequency
We used novel methods not previously assessed in apheresis donors to evaluate the potential
correlation with donation frequency. Evaluation of log(sTfR1/ferritin) demonstrates a
positive correlation with donation frequency (Fig. 1a). In addition, we observed a difference
in MCV and CHr between apheresis donors who donated 1–2 relative to those who donated
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>10 times (Figs. 1b and 1c). Furthermore, higher frequency donors were more likely to have
CHr < 32.6 pg (Fig. 1d) although the differences were not statistically significant. Our
findings demonstrate that apheresis donors exhibit relative ID and IRE, correlating with
donation frequency.

To validate our comparison of apheresis donors who donated 1–2 relative to those who
donated >10 times, we analyzed hepcidin/log ferritin. This measure of iron status
incorporates the evaluation of total iron stores (i.e., ferritin) as well as the more dynamic
regulation of iron availability for erythropoiesis and other iron-requiring physiological
processes (i.e., hepcidin) [28,29]. Hepcidin/log ferritin is higher in apheresis donors who
donated 1–2 relative to those who donated 3–10 donations and >10 times (Fig. 2a). This
finding suggests that the interrelatedness between ferritin stores and hepcidin response is
consistent with relative IRE in more frequent apheresis donors. Furthermore, correlations
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between hepcidin and donation frequency (Fig. 2b) and between serum ferritin and donation
frequency (Fig. 2c) suggest that increasing donation frequency negatively impacts iron
stores. An expected strong correlation between hepcidin and log ferritin was also observed
(Fig. 2d).

Correlation Between Platelet Count and Markers of Iron Restricted Erythropoiesis

To explore the association between thrombocytosis and ID, we analyzed the relationship
between markers of IRE or ID, and platelet count. As previously demonstrated [19,23], no
correlation between platelet count and serum ferritin (Fig. 3a) or hepcidin (Fig. 3b) was
found. However, we demonstrate a positive correlation between sTfR1 and platelet count
(Fig. 3c), confirming that platelet count increases progressively with IRE [25] and that
sTfR1 may be a useful marker of IRE in high frequency apheresis donors.
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DISCUSSION
Applying the definitions and reliable reference values of ID and IRE is complex and
dependent on the specifics of the population being analyzed. The main aim of this pilot
study was to evaluate novel markers of ID and IRE to identify the effect of frequent
apheresis donation and assess impact on platelet count in order to gain a greater

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understanding of their utility in this population. Furthermore, these parameters, as

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harbingers of anemia, provide information enabling donors and donor centers to identify risk
of potential ID after frequent apheresis donation, not traditionally associated with risk of ID.
Our control data for serum ferritin and TIBC correlate well with comparable individuals
within the NHANES database despite our relatively smaller sample size. Prior studies in
blood donor using serum ferritin concentration defined ID as serum ferritin <12 mg/dL, a
highly specific but insensitive indicator of ID, resulting in failure to identify ID in >30% of
cases [9,20–23,25,28]. Others used serum ferritin concentration between 22 and 40 mg/dL
[8,30] to better correlate with depleted bone marrow iron stores [10,11]. Our data
demonstrate a relative ID with a lower serum ferritin, hepcidin, iron, and transferrin
saturation in study donors relative to controls. The lack of difference in TIBC between study
donors and controls suggests that normal compensatory mechanisms result in normal
circulating iron despite relatively depleted iron stores, as expected from decreased hepcidin
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concentration. However, no differences in rates of absolute ID or iron depletion were noted

between study donors and controls. Furthermore, we demonstrate that donation frequency
correlates inversely with serum ferritin and hepcidin in study donors, both analyzed as
discrete and continuous measures of donation frequency, suggesting that increasing donation
frequency negatively impacts iron stores.

Although serum erythropoietin, hemoglobin, and reticulocyte count are no different in study
donors relative to controls, RBC count is increased. If anything, the borderline hemoglobin
suggests that study donors may have an increased total erythroid mass in circulation. We
analyzed markers of IRE. Evidence of IRE, without anemia, can be evaluated using MCV as
changes in cell size precede changes in hemoglobin concentration [31]. Furthermore,
reticulocyte hemoglobin concentration (i.e., CHr) has been previously reported as a useful
measure of recent changes in iron availability for erythropoiesis [9]. An additional indicator
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of early IRE is sTfR1 [32], and when combined with serum ferritin (i.e. log(sTfR1/ferritin))
provides a highly sensitive discrimination of clinically relevant IRE [9,11,12]. Although no
difference in MCV, CHr, and sTfR1are observed between study donors and controls, our
data demonstrate a relative increased in log(sTfR1/ferritin), indicating relative IRE in study
donors compared with controls.

Our findings also demonstrate decreased MCV and CHr in study donors who donate more
frequently and a correlation between donation frequency and log(sTfR1/ferritin), suggesting
that relatively more IRE occurs in more frequent apheresis donors. Although no parameter
was lower than previously suggested cutoffs for determining absent iron stores (in whole
blood donors [12]), more frequent donors were more likely to have CHr < 32.6 pg, again
suggesting decreased iron availability for erythropoiesis in more frequent apheresis donors
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as previously reported for RBC donors [33]. Because data for sTfR1 and log(sTfR1/ferritin)
are not standardized, comparison between assays and studies is unproductive. We report
results like several other studies using internal cutoffs, corresponding with 95th or 97.5th
percentile distribution in the study population to denote those with IRE [9,12].

More recently, circulating hepcidin concentration has been identified as the central negative
regulator of iron uptake, recycling, and availability [13]. A decreased serum hepcidin can
result from a decrease in iron stores or an increase in erythropoietin and expansion of

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erythroid mass [13,34]. Because ID results in erythroid expansion, it is possible that

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erythroid suppression of hepcidin indicates relative IRE. We have normalized hepcidin

concentration to log ferritin as a measure of hepcidin responsiveness to iron stores. This
enables us to evaluate the availability of iron for erythropoiesis. Our data demonstrate that
hepcidin/log ferritin correlates with donation frequency as a discrete and continuous
variable. Thus, our findings of decreased MCV and CHr in addition to hepcidin/log ferritin
provide evidence of the dynamic regulation of iron availability for erythropoiesis in concert
with total body stores in frequent apheresis donors, consistent with prior evidence
correlating hepcidin concentration with hemoglobin recovery in whole blood donors [28].
Methods for measuring erythroferrone, a newly described erythroid regulator of hepcidin
[34], are currently being developed to assess this important parameter.

Lastly, comparable with prior literature [7,9,28], we did not find a correlation between
platelet count and serum ferritin or hepcidin. However, we demonstrate for the first time a
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positive correlation between platelet count and serum sTfR1, confirming that platelet count
increases with progressive IRE [29] and suggesting that serum sTfR1 may be a useful
marker to stratify those apheresis donors who may benefit of iron replacement.

We are presenting data in some cases with what is defined as “intermediate strength”
correlations [26]. The intent of this delineation is to provide a broad perspective on the
meaning of correlation coefficients. As there may be strong correlations, with statistically
significant differences, that are not clinically meaningful, some intermediate strength
correlations are clinically important. Given the study sample composition and the interplay
of biological parameters, we present these statistically significant correlations in light of
their clinical importance. The expected strong positive correlation between log serum ferritin
and serum hepcidin [14] provides validity to this dataset.
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CONCLUSION
In conclusion, we evaluated iron- and erythropoiesis-related parameters in male apheresis
donors as a pilot study to understand the prevalence of ID, IRE, and their potential
correlation with donation frequency. We demonstrate that relative ID and IRE occur in study
donors who donated more frequently, and a positive correlation is evident between platelet
count and sTfR1. Our data also demonstrate that hepcidin/log ferritin provides the best
continuous measure of depleting iron availability for erythropoiesis; CHr and sTfR1 are also
clinically useful. Additional studies are needed to extend these findings to larger
populations, including younger, nonwhite, and female apheresis donors, and to establish
clinically useful cutoff measures to enable implementation in donor centers looking to tailor
iron replacement protocols to treat apheresis donors at risk for developing anemia as a
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consequence of frequent donation.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

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ACKNOWLEDGMENT
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The authors sincerely appreciate the volunteer donors who participated in our study, the efforts of Special Donor
Services and Collections staff who enabled the collection of this data, Tomas Ganz and Elizabeta Nemeth (UCLA)
for assistance with study design and data interpretation, and Jacobi Medical Center for sample analysis.

Contract grant sponsor: New York Blood Center funding to the Erythropoiesis Laboratory.

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Fig. 1.
Study donors exhibit iron restricted erythropoiesis which positively correlates with donation
frequency. (a) A positive correlation is observed in study donors and controls between
log(sTfR1/ferritin) and donation frequency (r = 0.3) with 2 study donors (and no controls)
with evidence of iron restricted erythropoiesis (open rhomboid). A lower MCV (b) and CHr
(c) are observed in study donors who have donated >10 donations in 12 months relative to
those who donated 1–2 donations (n = 8–31 donors per group). (d) Trend toward a higher
likelihood of CHr < 32.6 pg with increasing donation frequency (P = 0.14). (sTfR1, soluble
transferrin receptor 1; MCV, mean corpuscular volume; CHr, corpuscular hemoglobin of
reticulocytes).
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Fig. 2.
Severity of iron depletion in study donors correlates with donation frequency. (a) Lower
hepcidin/log ferritin can be observed in study donors who had donated platelets >10
donations relative to those who had donated 1–2 donations (n = 8–31 donors per group).
Inverse correlations (b) between hepcidin and number of donations and (c) between serum
ferritin and number of donations suggest negative impact of increasing donation frequency
on iron stores. (d) A confirmatory correlation between hepcidin and log ferritin was also
observed.
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Fig. 3.
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A positive correlation is observed between sTfR1 and platelet count in study donors. No
correlation is observed between serum ferritin (a) or serum hepcidin (b) and platelet count
although a correlation is observed between sTfR1 and platelet count (c). (sTfR1, soluble
transferrin receptor 1).

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TABLE I.

Study Participant Demographics

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Donor Control
Total number of participants 43 5
Average age ± SEM (years) 55 ± 1 54 ± 3 (NS)
Average number of donations in previous 12 months ± SEM 7±1 NA
Donors with non-plateletpheresis donations (%) 5 NA
Donors with history of low Hb/HCT deferral (%) 2 0 (NS)
Donors taking vitamins (%) 40 40 (NS)

SEM, standard error of the mean; NS, not statistically significant; NA, not applicable; Hb, hemoglobin; HCT, hematocrit; non-plateletpheresis
units, plasmapheresis or leukapheresis.
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TABLE II.

Iron-Related End Points in Study Donors and Controls

Li et al.

Study donor Control P values Normal range (males)

Serum ferritin (ng/mL) 55 ± 6 230 ± 86 <0.0001 12 – 300
TIBC(ug/dL) 347 ± 6 311 ± 21 0.11 250 – 370
Serum hepcidin (mg/mL) 33 ± 3 63 ± 11 0.001 29 – 254
Serum iron (ug/dL) 102 ± 4 139 ± 37 0.04 65 – 180
Transferrin saturation (%) 30 ± 1 45 ± 11 0.01 20 – 50
Fraction with ferritin < 22 ng/mL 0.2 0.0 0.57 NA
Fraction with ferritin < 40 ng/mL 0.3 0.2 0.65 NA

TlBC, total iron binding capacity; NA, not applicable.

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TABLE III.

Erythropoiesis-Related End Points in Study Donors and Controls

Li et al.

Study donor Control P values Normal range (males)

RBC count (106/uL) 4.9 ± 0.1 4.4 ± 0.4 0.04 4.2 – 5.7

Hemoglobin (g/dL) 14.8 ± 0.2 13.3 ± 1 0.05 13.1 – 17.3

MCV(fL) 93 ± 1 95 ± 4 0.32 80 – 96

CHr(pg) 31.5 ± 0.3 31.8 ± 1.6 0.56 a

23 – 34

Reticulocyte count (109/L) 71 ± 3 67 ± 14 0.71 30 – 130

Serum erythropoietin (mlU/mL) 12.3 ± 0.7 14.3 ± 2 0.41 0 – 19

Log(sTfR1/ferritin) 0.8 ± 0.03 0.6 ± 0.08 0.03 NA

RBC, red blood cell; MCV, mean corpuscular volume; CHr, corpuscular hemoglobin of reticulocytes; TfR1, transferrin receptor 1; sTfR1, soluble TfR1; NA, not applicable.
a
From Brugnara C, Schiller B, Moran J. Reticulocyte hemoglobin equivalent (Ret He) and assessment of iron-deficient states. Clin Lab Haematol 2006;28:303–308.

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