Blood, Vol. 92 No. 7 (October 1), 1998:
pp. 2590-2591
CORRESPONDENCE
How Old Are Dense Red Blood Cells? The Dog's Tale
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LETTER |
To the Editor:
Decades ago it was observed that young cohorts of radiolabeled human
red blood cells (RBC) became progressively enriched in the more dense
cell fractions as the cohorts aged.1 It was also shown that
the most dense cells were short-lived in circulation after reinfusion
into the donor.2 Consequently, density gradient centrifugation has served as the cornerstone methodology for the isolation and study of senescent human RBC. However, such studies have
not quantitatively assessed the degree of enrichment for old cells in
dense fractions, and the observed dispersion of RBC cohorts of any
particular age throughout the entire density gradient has been
bothersome to many.3
Quantitative studies of this issue in animals have generally
intensified the controversy. Experiments on hypertransfused rats have
shown that a progression of cells to higher densities (measured by mean
corpuscular hemoglobin concentration [MCHC]) occurs
early in the RBC life span, suggesting that density changes in rats are
associated primarily with maturation rather than
senescence.4 Similarly, hypertransfused mice have been
shown to display minimal age-associated changes across RBC density
fractions.5,6 Analysis of biotinylated rabbit RBC (B-RBC)
cohorts during their last 10 days of survival has shown an enrichment
of only twofold to threefold over unmarked (younger) cells in the most
dense fractions,7 and these senescent B-RBC comprise
only 
of the RBC in the dense fraction. Such results have
tended to raise questions regarding the validity of density
fractionation as a tool for isolating senescent RBC.
We have been evaluating RBC senescence in the dog based on the
conviction that the dog is a more appropriate model of human RBC
senescence than are small laboratory mammals. This assumption is
justified by strong similarities between human and dog RBC in important
features such as their comparatively long life spans (~110 to 120 days) and their low amounts of random RBC loss. We have validated the
biotinylation system for use in the dog and have shown that cells
greater than 104 days exhibit many characteristics of senescent human
cells (manuscript submitted), including increased autologous IgG binding.8 We have now used this unequivocal method of retrieving aged dog RBC to address two important questions: (1) at what cell age do RBC become a significant component of the most
dense fraction of cells? and (2) what is the approximate age
distribution of the 1% most dense cells in circulation? We predicted
that cells in all fractions would be ~100% biotinylated at day 1 and
that, with time, the percentage of nonbiotinylated cells would
increase, beginning in the lightest fraction and progressing to the
most dense fraction.
As shown in Fig 1, isolation of dog RBC on
arabinogalactan density gradients resulted in a crude cell age-based
separation, with younger unlabeled cells usually but not invariably
entering fractions of increasing density consecutively. Further,
unlabeled RBC entering circulation after biotinylation were generally
excluded from the most dense fraction for much of the RBC life span
(see hatched squares in Fig 1). Thus, dog 331 showed 
6%
nonbiotinylated cells in this fraction until after day 72, but
contained ~25% of the younger cells at day 86 (Fig 1A,
Table 1). Dog 794 showed 5%
nonbiotinylated cells until day 86 and wasn't significantly contaminated with younger cells until between days 93 and 100 (Fig 1B,
Table 1).
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| Fig 1.
Two mature male beagles were infused intravenously with N
hydroxysuccinimido (NHS)-biotin (35 mg/kg body weight in
dimethyl sulfoxide) to biotinylate all circulating RBC (99% each), as
described.9 One dog (794) was recovering from an idiopathic
anemia and had a low normal hematocrit of 37% at labeling that
increased to 52% by day 48, resulting in a partial cohort effect. The
other dog (331) was assessed to be completely normal. Blood was taken
for density gradient centrifugation at roughly 2-week intervals
initially and then weekly from day 86 to 105 to determine the
proportions of B-RBC in each density fraction (by flow cytometry) with
increasing mean cell age. Fraction 4 contains the densest 1% of RBC.
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Table 1.
Age Distribution and Purity of In Vivo Aged,
Biotinylated RBC in the Most Dense Fraction of Arabinogalactan
Fractionated Dog Cells
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The separations between days 72 and 105 allowed us to characterize the
approximate age distribution of the 1% most dense RBC (Table 1). For
instance, analysis of dog 331 cells at postbiotinylation day 86 showed
that the most dense fraction contained 76% biotinylated RBC,
indicating that only 24% of the RBC were younger than 86 days old. A
sharper age dependency was obtained when a younger cohort of cells was
biotinylated, as with dog 794, where the most dense fraction was 95%
B-RBC at the same time point. Even at day 93, the densest fraction of
dog 794 RBC was still 84% B-RBC, demonstrating a remarkable enrichment
of old RBC in this highly dense fraction.
Our results clearly provide greater justification for use of density
gradient centrifugation for isolation of senescent RBC than previous
studies using rabbit biotinylated cells have indicated.7 At
least part of the difference between the two studies may be due to the
different methods used for identifying B-RBC. The purity of the
rabbit's most dense fractions was likely underestimated, because the
bead-binding assay used in that study is much less sensitive than flow
cytometry to the presence of the biotin label.9 Also, the
high percentage of random RBC removal in the rabbit and most
rodents10,11 raises questions whether the senescent cell
recognition mechanism is similar to humans and dogs in these smaller species. Finally, differences in the centrifugation media (Percoll-Hypaque v arabinogalactan) could have significantly
affected the results, because in our hands arabinogalactan gradients
yield a higher percentage of senescent cells in the dense fraction than do Percoll gradients.
In summary, these results confirm that the 1% most dense RBC from
dogs represent a predominantly aged population of cells. Labeling a
cohort of young cells further enhances the enrichment for old RBC in
this fraction. To the extent that canine RBC serve as an accurate model
for human RBC, it can be concluded that the changes that occur late in
the human RBC life span and trigger RBC removal can be studied by
fractionating the cells on arabinogalactan density gradients.
John A. Christian
Jiazhen Wang
Nadya Kiyatkina
Department of Veterinary
Pathobiology
Michael Rettig
Philip S. Low
Department of Chemistry
Purdue University
West
Lafayette, IN
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ACKNOWLEDGMENT |
This work was funded in part by National Institutes of Health
Grant No. GM24417.
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