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RED CELLS
From the University of Cincinnati College of Medicine,
Children's Hospital Research Foundation, and Cincinnati Comprehensive
Sickle Cell Center, Cincinnati, OH.
Sickle red blood cells (RBCs) become depleted of potassium,
leading to dehydration and abnormally elevated cellular density. The
increased sickling that results is important for both hemolysis and
vasocclusion. In this study, sickle cells were subjected to high-speed centrifugation, and the bottom 15% were isolated. This procedure removed light cells and to a variable degree enriched cells
that were denser than normal to produce a high-density-enriched (HDE)
population of sickle cells. Autologous HDE cells from 3 subjects were
labeled with biotin and re-infused. The following determinations were
performed: (1) the survival and density changes of HDE cells; (2) the
amount of fetal hemoglobin (HbF) in labeled cells after magnetic
isolation; (3) the percentage of labeled F cells; (4) the percentage of
labeled cells displaying external phosphatidylserine (PS). For patients
with 3.5%, 4.5%, and 24% HbF in the HDE RBCs, the circulation
half-time was 40, 80, and 180 hours, respectively. The percentage of
HbF (measured in all 3 subjects) and of F cells (measured in 2 subjects) in labeled RBCs increased with time after re-infusion,
indicating that HDE F cells have longer in vivo survival than HDE non-F
cells. The percentage of PS+, biotin-labeled HDE cells
showed no consistent increase or decrease with time after re-infusion.
These data provide evidence that HDE sickle cells, especially those
that do not contain HbF, have a very short in vivo survival, and that
the percentage of PS+ cells in a re-infused HDE population
does not change in a consistent manner as these cells age in the circulation.
(Blood. 2000;96:3610-3617) Sickle red blood cells (RBCs) tend to
become potassium depleted and thus dehydrated and abnormally
dense. The higher concentration of sickle cell hemoglobin (HbS) leads
to more polymerization and sickling, and contributes to both hemolysis
and vasocclusion. Several interacting cation transport pathways mediate
dehydration, including (1) a nonselective increase in cation
permeability related to morphological sickling, (2) a calcium-dependent
K channel (Gardos) that most likely depends on calcium influx through
the nonselective sickling-dependent pathway, and (3) KCl cotransport, a
normal pathway in immature erythroid cells that is very active in
sickle RBCs, owing at least in part to their young age distribution but perhaps also to a specific activation related to the presence of
HbS.1,2 Administration of clotrimazole,3 an
inhibitor of the Ca++-dependent K channel, or
magnesium,4 which inhibits KCl cotransport, leads to
partial normalization of sickle RBC hydration.
When sickle RBCs are separated by density, there are many
cells with normal density, but also a variable number of light and dense cells. The number of dense RBCs varies widely among patients but
in the absence of painful episodes is relatively constant for each
patient.5 Young sickle cells, including reticulocytes and
the even less mature cells displaying transferrin receptor, are seen
not only in the light fractions as expected, but also in the dense
fractions. Both the lightest and densest cells contain less fetal
hemoglobin (HbF) than cells with moderate density,6 and
there is evidence that only cells lacking HbF can become extremely dehydrated soon after leaving the marrow.7 Previous
studies have used several direct and indirect techniques to show that sickle cells containing HbF survive better in the
circulation.6,8-10
In general, dense sickle cells tend to have more severe membrane
abnormalities when compared with cells with normal
hydration.11 Recently, it has been shown that a higher
percentage of cells in the dense fraction have lost normal phospholipid
asymmetry and display phosphatidylserine (PS) on the outer membrane
leaflet.12 It has been postulated that these cells, owing
to the exposure of PS, may be thrombogenic and readily recognized and
removed by macrophages.
In the current studies, high-density-enriched (HDE) sickle RBCs
were labeled with biotin, re-infused, and analyzed in subsequent blood
samples. The biotin label has recently been shown by us10 and others13 to be equivalent to a 51Cr label
in the measurement of human RBC survival. In addition to survival
characteristics, the current studies also examined postinfusion changes
in the hydration state of HDE cells, differences between the behavior
of HDE F cells and non-F cells, and changes in PS exposure as the cells
aged in vivo.
Approximately 90 mL blood was drawn into
heparinized (15 U/mL) syringes with informed consent. Dense RBC
enrichment, biotin labeling, and re-infusion were performed within
approximately 6 hours. All studies were autologous, and the subjects
had neither been transfused for at least 3 months nor had a recent
painful episode.
Red cells were washed 3 times with at least 2 volumes of Dulbecco's
phosphate-buffered saline (D-PBS; Gibco BRL, Grand Island, NY).
After the last centrifugation, the packed RBCs were transferred to
gas-sterilized, polypropylene, high-speed centrifuge tubes (16 × 98
mm) and spun (18 000g maximum, 22°C) for 90 minutes. The top 85% of the red cell column was carefully removed and
discarded. The bottom 15% of the red cell column (HDE cells, 2 to 3 mL) was labeled with biotin as previously described,10
with the use of 3 µg/mL NHS-biotin. Prior to re-infusion, a small
aliquot of the labeled RBCs was reacted with streptavidin-phycoerythrin
(SA-PE) and analyzed by flow cytometry10 to ensure that all
the cells were labeled and that there was baseline separation of
labeled and unlabeled cells on the flow cytometric histogram. The
remaining biotin-labeled RBCs were re-infused through an 18-µ blood
filter (Hem-O-Nate; Bard, Salt Lake City, UT).
Postinfusion blood samples were typically obtained at 7.5, 10, 12.5, 15, and 20 minutes; 1, 2, 4, 8, 16, and 24 hours; 2 and 3 days, and as
required thereafter. To facilitate blood sampling, the subjects were
admitted for 1 day to the Clinical Research Center at the University of
Cincinnati Medical Center. The percentage of labeled RBCs was measured
at each time point,10 and the survival of the HDE cells
determined. For selected postinfusion time points, the RBCs were
separated into 6 density fractions as described,10 and the
number of labeled cells in each fraction was determined. For the same
selected samples, labeled RBCs were isolated by means of
streptavidin-coated magnetic beads (Dynal, Lake Success, NY), and the HbF content was determined by high-performance liquid chromatograhy (HPLC) as previously described.10 In
addition, these samples were analyzed with 2-color flow cytometry for
biotin and HbF. After incubation with SA-PE, the cells were fixed,
permeabilized, and incubated with a fluorescein isothiocyanate
(FITC)-conjugated monoclonal antibody to HbF (Wallac; Akron,
OH).14 A 2-color flow cytometric protocol was used to
determine the percentage of F cells both in the unlabeled cells, which
represent the entire circulating cell population, and in the labeled
cells, which represent the re-infused cells as they age in the circulation.
To investigate changes in externalized PS as the labeled HDE cells
aged, blood samples were incubated first with SA-PE and then with
Annexin V-FITC (R&D Systems; Minneapolis, MN) according to the
manufacturer's protocol, with the use of 1 µg/mL Annexin V-FITC and
108 RBCs per milliliter. The cells were analyzed by 2-color
flow cytometry, allowing determination of the percentage of
PS+ cells in the labeled and unlabeled populations.
At selected postinfusion time points, RBCs from density
fraction 2 (1.073 < In vivo survival of density-enriched sickle cells
Longer survival of F cells in the HDE population
The amount of F per F cell can be estimated either directly or
indirectly. Dover et al8 used a direct microscopic
immunodiffusion method to show that sickle erythrocytes had 10.9 ± 3.2 (1 SD) pg of HbF per F cell. Horiuchi et al17 used a
direct fluorescent image analysis technique to determine a range of 2.7 to 12.8 in 5 patients with sickle cell disease. In the indirect method,
2 measurements are made: the percentage of HbF in all the cells and the
percentage of F cells. It is assumed that all the HbF is in the F
cells, and the HbF per F cell is calculated as follows: HbF/Fcell = (% HbF/% F cells) · MCH, where MCH is mean
corpuscular hemoglobin. When this formula is applied to the data of
Dover et al8 with a value of 31 for MCH, an average of 12 pg HbF per F cell is obtained, which agrees well with the directly
measured value.
For each time point in Figure 3 (patients H7-2 and H7-3), the HbF per F
cell was calculated as described above for the unlabeled and labeled
cells. The values for HbF per F cell (Figure
5) show no change with time in the
labeled HDE cells. In fact, at all times, HbF per F cell was
essentially the same for the labeled HDE cells and the unlabeled cells,
which include cells with all density values. Values of HbF per F cell
represent averages for the population measured, but presumably there is
a range of values within each population. If there is selection within
the F-cell population (ie, if F cells with higher values of HbF per F
cell survive longer than F cells with lower values), then HbF
per F cell for the biotin-labeled HDE cells would most likely be higher (since they are older on average) and would increase further as the
cells aged in the circulation. These differences were not observed,
implying a lack of selection within the F-cell population.
The time-dependent enrichment of F cells in the circulation indicates
that HDE F cells survive longer than HDE non-F cells. As shown
previously,10 individual survival curves for these 2 cell
types can be derived from the overall survival data and the percentages
of F and non-F cells at each time point. These percentages may be taken
directly from the flow cytometric assays or calculated from the
percentage of HbF (by HPLC) and the HbF per F cell for each patient.
Figure 6 shows the results of this analysis. For all subjects, the non-F cells had a shorter survival and
appeared to have a biphasic disappearance, whereas the F cells had a
longer survival and a linear disappearance.
Time-dependent changes in the percentage of
PS+ sickle RBCs
Valinomycin resistance of the older, light, labeled cells
A large majority of older, light, labeled RBCs are
valinomycin-resistant (Table 1),
consistent with Na+ rather than K+ as the
dominant monovalent cation. From these data, it may be concluded that
the majority of older cells in the light fraction are resistant to the
dehydrating effect of valinomycin.
The studies presented here examine the time-dependent in
vivo behavior of a subpopulation of denser sickle cells. The focus was
on factors either known to influence cellular survival characteristics (dehydration, HbF) or suspected of doing so (PS exposure).
Understanding the relationships between RBC survival, PS
externalization, HbF content, and dehydration is a difficult task,
which has become increasingly important as therapeutic agents that
modify one or more of these cellular properties become available. The
clinical use of hydroxyurea to increase HbF and the emergence of
experimental agents that change in vivo sickle RBC hydration
state3,4 have underscored the gaps in understanding sickle
cell pathophysiology. The studies presented here apply cell-by-cell
flow cytometric analysis techniques to study the changes that occur in
HDE cells as they age in the circulation. These longitudinal studies
give another dimension to the study of sickle cells, since they allow the study of time-dependent processes in vivo. When interpreting these studies, one should keep in mind that the labeled HDE cells are highly selected, both in vivo prior to the study and in
vitro as part of the study. The rate of dehydration undoubtedly varies among sickle cells, but there is evidence that most cells become dehydrated early in their lifespan.6,10 Since the young
light cells are removed during the isolation of HDE cells, changes that occur early in the life of a sickle cell would most likely not be
apparent in these studies.
The short survival of dense sickle cells in one patient was first
described by Bertles and Milner6 in 1968 and is confirmed in the current studies. The time for removal of 50% of the HDE cells
ranged from 40 to 180 hours. For non-F cells, removal from the
circulation appeared to occur in 2 phases, with the first phase
(approximately 70% of non-F cells) having a very short 50% survival of 40 to 80 hours. The remaining non-F cells (30%)
survived about the same amount of time as F cells for each subject,
which ranged from approximately 120 (H7-1) to 330 (H7-2 and H7-3)
hours. These results are summarized in Table
2. The shorter F-cell survival in subject
H7-1 may be due to the greater percentage of HDE cells in the dense
fractions 5 and 6. These survival times are much shorter than for
unfractionated sickle cells, which had corresponding 50% survival
values of 240 to 400 hours for all cells, 120 to 170 hours for non-F
cells, and 350 to 600 hours for F cells.10
The 2-phase disappearance of non-F cells was a consistent finding in these patients and was present in both methods of HbF analysis. Examination of the analogous survival curves for unfractionated cells10 shows that there was some evidence of a 2-phase removal but that it was less striking. One possibility is that the 2 phases are a reflection of the different behavior of the intermediate and dense cells present in the labeled population. However, this is not supported by patient H7-1, who had a much purer population of dense cells but the same 2-phase survival behavior. The remarkably stable number of light, labeled RBCs in fractions 1 and 2 are of considerable interest. These cells do not behave as expected for a typical, young light cell that was labeled and re-infused along with the HDE cells. First, a young light cell would be expected to quickly move to the denser fractions. Indeed, in studies with unfractionated cells,10 the number of labeled light cells declined markedly in the first 2 days after re-infusion. Second, after any initial changes the number of light cells was stable as long as the labeled HDE cells remained in the circulation in these studies and for at least 35 days with unfractionated cells.10 If these cells were light at the time of labeling and remained light, some decrease in their number would be expected at later times, even if they had a relatively long survival. We hypothesize that the most likely explanation for these cells is a steady conversion of dense cells to light cells prior to removal from the circulation. An unequivocal test of this hypothesis, however, will require the re-infusion of a pure population of dense cells, so that the appearance of light cells unambiguously demonstrates their formation from denser cells. Most of the older (at least 8 days old), labeled light cells were resistant to dehydration by the potassium ionophore valinomycin, while only a low percentage of the unlabeled cells in the same fraction were resistant. Bookchin et al15,16 showed that a small fraction of light sickle cells that were not reticulocytes resisted the dehydrating effect of valinomycin and contained high sodium and low potassium. In the current work, we show that older cells (well beyond the reticulocyte stage) are present in the light fraction at an essentially constant number after re-infusion of labeled cells. These older, labeled, light cells were also shown to be resistant to valinomycin, although cation content was not directly measured. Valinomycin resistance does not prove that the older light cells contain high sodium and low potassium, since other possibilities (eg, severe inhibition of conductive chloride transport) may exist. However, it seems likely that the labeled, older, valinomycin-resistant cells would show the same sodium loading and potassium depletion as the unlabeled, nonreticulocyte, valinomycin-resistant cells demonstrated in the studies of Bookchin et al.15,16 High sodium content in older light cells implies a loss of volume regulation, confirming that they are not typical light cells and supporting the concept that they represent a terminal state. HbF in the labeled cells was measured in 2 ways as they aged in the circulation. In the first method, biotinylated cells were isolated magnetically and HbF was determined by HPLC. In the second method, the percentage of HbF-containing cells in the biotin-positive population was determined by flow cytometry. These 2 methods of analysis of HbF-containing cells are equivalent only if there is no change in the amount of HbF per F cell as the cells age and are removed from the circulation. That is, if there is a range of HbF in F cells, and if the cells with higher F per F cell survive longer, then the F per F cell of the remaining F cells will be higher, and the amount of HbF in the magnetic bead samples will increase faster than the percentage of F cells. On the other hand, if the F-cell population is essentially uniform, at least with respect to survival, then the ratio of HbF content and percentage of F cells would remain constant, as observed in these experiments. Nevertheless, these experiments with HDE cells do not rule out the possibility that such selection takes place at a very young cell age, eg, during the reticulocyte stage or soon afterward. The data of Dover et al8 showed lower HbF per F cell in sickle reticulocytes compared with mature sickle cells, supporting a survival advantage for F cells with higher levels of HbF per F cell. Our data suggest that this selection does not extend to older, denser cells. It has previously been shown that the percentage of PS+
cells in the very light and dense fractions is higher than the
percentage in the middle fractions.12 The presence of
older and perhaps damaged cells in the light fractions, as discussed
above, could contribute to the higher percentage of PS+
cells in those fractions. The day-to-day variability observed for the
unlabeled cells in the current studies is consistent with the data of
Wood et al,18 who showed that individual patients often
have marked variation in the percentage of PS+ cells.
Analysis of the survival of PS+ cells with the use of the
biotin label is made more difficult by the possibility that labeled
cells that were PS In summary, these studies show that HDE sickle cells have a very short survival in the circulation, and that HbF provides a significant advantage in this dehydrated population of cells. Furthermore, HDE cells show no consistent change in PS positivity as they age and are selected in the circulation, implying that the survival of PS+ is not extremely short. Finally, an older population of light, labeled cells was detected, supporting the hypothesis that these cells are derived from the dense cell population and represent a terminal state.
Submitted September 7, 1999; accepted July 11, 2000.
Supported by National Institutes of Health grants RO1 HL51174, RO1 HL57614, and P60 HL58421.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Robert S. Franco, University of Cincinnati College of Medicine, Vontz Center for Molecular Studies, Hematology/Oncology Division ML#508, 3125 Eden Ave, Cincinnati, OH 45267-0508; e-mail: robert.franco{at}uc.edu.
1.
Oliviero O, Vitoux D, Galacteros F, et al.
Hemoglobin variants and activity of the (K+Cl 2. Vitoux D, Beuzard Y, Brugnara C. The effect of hemoglobin A and S on the volume- and pHdependence of K-Cl cotransport in human erythrocyte ghosts. J Membr Biol. 1999;167:233[Medline] [Order article via Infotrieve]. 3. Brugnara C, Gee B, Armsby CC, et al. Therapy with oral clotrimazole induces inhibition of the Gardos channel and reduction of erythrocyte dehydration in patients with sickle cell disease. J Clin Invest. 1996;97:1227[Medline] [Order article via Infotrieve]. 4. De Franceschi L, Bachir D, Galacteros F, et al. Oral magnesium supplements reduce erythrocyte dehydration in patients with sickle cell disease. J Clin Invest. 1997;100:1847[Medline] [Order article via Infotrieve].
5.
Ballas SK, Smith ED.
Red blood cell changes during the evolution of the sickle cell painful crisis.
Blood.
1992;79:2154 6. Bertles JF, Milner PFA. Irreversibly sickled erythrocytes: a consequence of the heterogeneous distribution of hemoglobin types in sickle-cell anemia. J Clin Invest. 1968;47:1731.
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Franco RS, Barker-Gear R, Miller MA, Williams SM, Joiner CH, Rucknagel DL.
Fetal hemoglobin and potassium in isolated transferrin receptor-positive dense sickle reticulocytes.
Blood.
1994;84:2013 8. Dover GJ, Boyer SH, Charache S, Heintzelman MS. Individual variation in the production and survival of F cells in sickle-cell disease. N Engl J Med. 1978;299:1428[Abstract].
9.
Franco RS, Thompson H, Palascak M, Joiner CH.
The formation of transferrin receptor-positive sickle reticulocytes with intermediate density is not determined by HbF content.
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1997;90:3195 10. Franco RS, Lohmann J, Silberstein EB, et al. Time-dependent changes in the density and hemoglobin F content of biotin-labeled sickle cells. J Clin Invest. 1998;101:2730[Medline] [Order article via Infotrieve].
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Hebbel RP.
Beyond hemoglobin polymerization: the red blood cell membrane and sickle disease pathophysiology.
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1991;77:214 12. de Jong K, van Rossum L, Lubin BH, Styles SA, Kuypers FA. Phosphatidyserine externalization in subpopulations of sickle cells [abstract]. Blood. 1997;90:125a. 13. Mock DM, Lankford GL, Widness JA, Burmeister LF, Kahn D, Strauss RG. Measurement of red cell survival using biotin-labeled red cells: validation against 51Cr-labeled red cells. Transfusion. 1999;39:156[Medline] [Order article via Infotrieve]. 14. Campbell TA, Ware RE, Mason M. Detection of hemoglobin variants in erythrocytes by flow cytometry. Cytometry. 1999;35:242[Medline] [Order article via Infotrieve]. 15. Bookchin RM, Etzion Z, Sorette M, Mohandas N, Lew VL. Detection of a low-density, low-K+, non-reticulocyte subpopulation of sickle cell anemia (SS) and normal (AA) red cells (RBCs) [abstract]. Blood. 1995;86:473a.
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Bookchin RM, Etzion Z, Sorette M, Mohandas N, Skepper JN, Lew VL.
Identification and characterization of a newly recognized population of high-Na+, low-K+, low-density sickle and normal red cells.
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2000;97:8045 17. Horiuchi K, Osterhout ML, Kamma H, Bekoe NA, Hirokawa KJ. Estimation of fetal hemoglobin levels in individual red cells via fluorescence image cytometry. Cytometry. 1995;20:261[Medline] [Order article via Infotrieve].
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© 2000 by The American Society of Hematology.
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  K. I. Ataga, W. R. Smith, L. M. De Castro, P. Swerdlow, Y. Saunthararajah, O. Castro, E. Vichinsky, A. Kutlar, E. P. Orringer, G. C. Rigdon, et al. Efficacy and safety of the Gardos channel blocker, senicapoc (ICA-17043), in patients with sickle cell anemia Blood, April 15, 2008; 111(8): 3991 - 3997. [Abstract] [Full Text] [PDF]
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 F. A. Kuypers Membrane Lipid Alterations in Hemoglobinopathies Hematology, January 1, 2007; 2007(1): 68 - 73. [Abstract] [Full Text] [PDF]
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 R. M. Bookchin, Z. Etzion, N. Daw, T. Tiffert, and V. L. Lew Generation of the High-Na+, Low-K+ Red Blood Cell (RBC) Fraction of Normal and Sickle Cells - A Possible Terminal State. Blood (ASH Annual Meeting Abstracts), November 16, 2006; 108(11): 1243 - 1243. [Abstract] [PDF]
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R. S. Franco, Z. Yasin, M. B. Palascak, P. Ciraolo, C. H. Joiner, and D. L. Rucknagel The effect of fetal hemoglobin on the survival characteristics of sickle cells Blood, August 1, 2006; 108(3): 1073 - 1076. [Abstract] [Full Text] [PDF]
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Z. Yasin, S. Witting, M. B. Palascak, C. H. Joiner, D. L. Rucknagel, and R. S. Franco Phosphatidylserine externalization in sickle red blood cells: associations with cell age, density, and hemoglobin F Blood, July 1, 2003; 102(1): 365 - 370. [Abstract] [Full Text] [PDF]
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J. D. Holtzclaw, M. Jiang, Z. Yasin, C. H. Joiner, and R. S. Franco Rehydration of high-density sickle erythrocytes in vitro Blood, September 26, 2002; 100(8): 3017 - 3025. [Abstract] [Full Text] [PDF]
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K. de Jong, R. K. Emerson, J. Butler, J. Bastacky, N. Mohandas, and F. A. Kuypers Short survival of phosphatidylserine-exposing red blood cells in murine sickle cell anemia Blood, September 1, 2001; 98(5): 1577 - 1584. [Abstract] [Full Text] [PDF]
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K. de Jong, S. K. Larkin, L. A. Styles, R. M. Bookchin, and F. A. Kuypers Characterization of the phosphatidylserine-exposing subpopulation of sickle cells Blood, August 1, 2001; 98(3): 860 - 867. [Abstract] [Full Text] [PDF]
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| Copyright © 2000 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
< 1.083) were incubated with
valinomycin, a potassium-specific ionophore. Cells with a normal high
concentration of potassium will become dehydrated when incubated
in the presence of valinomycin, whereas those containing sodium as the
dominant monovalent cation will remain hydrated. Valinomycin exposure
was as described by Bookchin et al.
, F cells; 
, non-F cells. For the panels on the left, the
survival for each cell type was derived from the overall survival, the
percentage of HbF in the magnetically isolated labeled cells, and the
HbF per F cell for each patient. For the panels on the right, the
overall survival and the flow cytometric quantitation of the percentage
of labeled F cells were used in a similar manner. Formulas and sample
calculations are given in Franco et al.
at the time of re-infusion could
become PS+ as they age in the circulation. However, the
data do put some limits on the survival characteristics of
PS+ cells. If a discrete population of HDE cells
experiences irreversible loss of asymmetry prior to labeling, and if
these cells have a very short survival in the circulation, then we
would expect the percentage of biotin-labeled PS+ HDE cells
to decrease markedly shortly after re-infusion. This was not observed.
On the other hand, if PS positivity is a more dynamic process, with
cells regaining asymmetry, or with a balanced removal and formation of
PS+ cells during the aging process, then the percentage of
PS+ cells would be more stable, as seen in these
experiments. However, even in this instance, the circulation time of
PS+ cells could not be extremely short, since the
replacement of labeled PS+ cells by conversion of labeled
PS