From www.bloodjournal.org by guest on April 1, 2017. For personal use only.
Improved Isolation of Normal Human Reticulocytes Via Exploitation of
Chloride-Dependent Potassium Transport
By Martin P. Sorette, Kathleen Shiffer, and
Margaret R. Clark
Studies on normal human reticulocytes have been limited by
a lack of methods for effective reticulocyte enrichment. This
study shows a convenient new approach for selective enrichment of reticulocytes from normal blood samples. We have
developed a modified arabinogalactan density gradient that
contains high potassium levels, approximating the internal
cation composition of red blood cells (RBC). The low-density
populationsfrom this gradient are enriched in reticulocytes,
and the highly selected lowest density fraction shows a much
higher reticulocyte enrichment than that obtained with high
sodium chloride arabinogalactan density gradients, or other
previously reported density gradient methods. We found
that this improved isolation is caused by suppression of
potassium loss and reticulocyte dehydration via chloride
(KCI) cotransport.When the low-density fraction of RBC from
a high-potassium gradient was subsequently incubated in
high sodium chloride medium and reseparated oli a sodium
chloride density gradient, the reticulocytes dehydrated and
were recovered in high-density fractions. The highestdensity fractions from this secondary gradient yield 95% to
99% reticulocytes. We anticipate that this method will benefit investigatorswho require reticulocyte enriched populations for a wide variety of applications.
o 1992by The American Society of Hematology.
S
have reticulocyte counts exceeding those previously reported for other density gradient methods. Subsequent
incubation and secondary separation of these fractions in
high-sodium (Na) chloride medium yields reticulocyte enrichment comparable to that attained by affinity-isolation
techniques.
TUDIES ON human reticulocytes, which constitute
approximately 0.5% to 1.5% of red blood cells (RBCs)
in normal adult individuals, have been limited by the
difficulty of isolating pure populations from normal blood.
Since the observation by Key1 in 1921 that reticulocytes are
more bouyant than erythrocytes, many studies have used
both density-based and cell size-based separation methods
to prepare reticulocyte enriched fractions. Centrifugation
of peripheral blood on density gradients of bovine serum
albumin* and arabin~galactan~
under isopycnic equilibrium
conditions, and sedimentation velocity separations using
phthalate esters: Ficoll: Renografin,6 Percoll,7 combined
Percoll-Renografin6 and Percoll-arabin~galactan~
have been
used both for erythrocyte separation and reticulocyte enrichment. In some studies, highly purified populations of
reticulocytes have been obtained from patients with hemolytic anemia whose whole blood reticulocyte counts may
average 15% to 20%. When used to separate normal human
blood, however, these methods produced maximal reticulocyte enrichment of only 5% to 20% reticulocytes. Because
of the relatively poor resolution of reticulocytes on density
gradients, other isolation techniques have been developed
to separate reticulocytes from erythrocytes based on their
differing surface characteristics. In one study that used
differential lectin-mediated agglutination of erythrocytes, a
14-fold enrichment of reticulocytes from a normal blood
sample was reported.1° Affinity chromatography on transferrin-sepharose” and immunomagnetic separation using a
monoclonal antibody to the transferrin receptor12 resulted
in preparations from normal individuals with reticulocyte
counts between 23% to 35% and 98% to loo%, respectively.
Here we describe a rapid, simple method for density
separation of normal human blood on discontinuous arabinogalactan gradients that results in selective enrichment of
reticulocytes. This method is based on the differential
bouyant density of reticulocytes in an isotonic highpotassium (K) medium that approximates the internal
cation content of human RBCs. We hypothesized that
chloride (C1)-dependent K loss from reticulocytes might
alter their hydration state during density gradient separation. By suppressing K loss, we obtained highly selected
low-density RBC populations from high-K gradients that
Blood, Vol80, No 1 (July 1). 1992: pp 249-254
MATERIALS AND METHODS
Chemicals and reagents. Arabinogalactan (Stractan 11) was
obtained from the St Regis Paper Co (Tacoma, WA). Ethidium
Bromide, EGTA (Ethyleneglycol-bis-(p-amino-ethyl ether) N,N’Tetra-Acetic Acid), EDTA (ethylenediaminetetraacetic acid), microcrystalline cellulose (Sigmacell type 50) a-cellulose, bovine
serum albumin, inosine, adenine, glucose and penicillinlstreptomycin were purchased from Sigma Chemical Co (St Louis, MO).
HEPES (N-2-hydroxyethylpiperazine-N‘-2-ethane
sulfonic acid),
HEPES Na salt, and PIPES (piperazine-N,N-bis (2ethanesulfonic
acid) were products of Calbiochem (La Jolla, CA). New Methylene
Blue N (Brecher formula) was purchased from J.T. Baker Chemical GJ(Phillipsberg, NJ). Reagent grade Na chloride, Na nitrate, K
chloride, and magnesium chloride were purchased from Fisher
Chemical Co (Fair Lawn, NJ).
Collection of blood samples. After obtaining informed consent
under a protocol in accordance with the principles of the Helsinki
Declaration and approval by the Human and Environmental
From the Department of Laboratory Medicine and the Cancer
Research Institute, University of Califomia, San Francisco; and Bio
Rad Inc, Hercules, CA.
Submitted September 24, 1991; accepted March 5, 1992.
Supported by Grants from the US Public Health Service No. DK
32095 and HL 20985.
This ispublication No. 128from the MacMillan-CargillHematology
Research Laboratory.
A preliminary report of this study has been presented in abstract
form:Sorette MP, Clark MR: Improved Isolation of Normal Reticulocytes on Modified Arabinogalactan Density Gradients. Blood (Suppl
I ) , 1991.
Address reprint requests to Margaret R. Clark, PhD, Box 0905,
University of Califomia, San Francisco, CA 94143.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
0 1992 by The American Society of Hematology.
0006-4971/92/8001-0016$3.00/0
249
From www.bloodjournal.org by guest on April 1, 2017. For personal use only.
SORETTE, SHIFFER, AND CLARK
250
Density (g/mL)
Na
K
1.0785
1.0829
1.0880
1.0919
1.0964
1.1 009
1.1 055
1.1102
1.1148
1.1 195
Protection Committee of the University of California, San Francisco, venous blood was collected from healthy normal volunteers
into EDTA or acid-citrate-dextrose (ACD) (Vacutainer; Becton
Dickinson, Mountain View, CA). Leukocytes and platelets were
removed by filtration through a column of ~ e l l u l o s e . ~ ~ ~ ~ ~
Arabinogalactan density gmdienrs. Arabinogalactan (Stractan
11) was decolorized using magnesium oxideIs and activated charcoal and purified according to Corash? For initial experiments at
pH 7.4, stock arabinogalactan solutions were prepared that, based
on available water, contained HEPES (20 mmollL), MgC12 (1
mmol/L), NaH2P04(1 mmol/L), glucose (IO mmollL), EGTA (0.5
mmol/L), adenine (2 mmollL), inosine, (12 mmollL), penicillin
(100 UlmL), streptomycin (100 pglmL). NaCl gradients contained
KCI (IO mmollL), NaCl (114 mmollL), and KCI gradients contained NaCl (12 mmol/L), KCI (115 mmollL) added to the stock
solution. The pH was adjusted to 7.4, and the osmolality to 290
mOsm using a vapor pressure osmometer (Model 5100 C; Wescor
Inc, Logan, UT). For subsequent experiments, NaNO3 and NaCl
gradients contained PIPES, pH 6.8 (20 mmollL). glucose (IO
mmollL), adenine (2 mmollL), inosine (12 mmollL), ouabain (0.2
mmollL), piretanide (0.1 mmol/L), and either NaNO3 (165
mmollL) or NaCl (150 mmol/L). The pH was adjusted to 6.8 and
the osmolality to 290 mOsm. Discontinuous gradients were prepared by dilution of stock Stractan with isosmolar buffers of the
same composition, using nine layers ranging in equal steps from
1.083 to 1.124 glmL density. These were layered in 17 mL tubes
onto a cushion of at least 1.133 glmL density. The gradients were
centrifuged in a swinging bucket rotor in an ultracentrifuge
(Beckman W-50)
for 45 minutes at 74,oOg at 20°C. The separated
cells were collected from the gradient interfaces using a Pasteur
pipet, duringwhich the walls of the tube were washed with buffer to
avoid contamination of subsequent layers. The separated cells
were washed three times to remove residual Stractan and were
resuspended to a defined volume.
RBCs and mticulqte density dishburion. RBC distribution in
the density gradient was determined by counting the cells in each
gradient population with an electronic cell counter (Model Zel;
Ffgl. D.nritydbtributionofRBCfromomindMdual in high NaCl and KCI gradients, pH 7.4. The
gradients are identical with respect to density. Note
that there is a shift t o lower densities in the K
gradient compared with the Na gradient. A distinct
population of low-density, reticulocyte-enrichedREC
(indicated by arrow) is clearly isolated in the high-K
gradient. This population is markedly reduced in the
high-Na gradient.
Coulter Electronics, Hialeah, FL). Mean cell hemoglobin concentration (MCHC) was determined from a spun hematocrit and
spectrophotometric measurement of hemoglobin as the cyanomethemoglobin complex. Manual reticulocyte counts were made on
1,OOO cellslsample stained with New Methylene Blue N.16 Reticulocyte counts by flow cytometry were performed on ethidium bromide stained samples1' using the Becton Dickinson Facscan flow
cytometer (Beckton Dickinson, Mountain View, CA). Gating on
the light-scattering signal was used to discriminate RBC from
particulate debris. Fluorescence was measured with excitation at
488 nm provided by an argon ion laser and detected through
narrow-band emmission filters at 530 nm (FLl, Green) or 585 nm
(FU, Red). The fluorescence measurements were standardized
using fluorescent microspheres (Calibrite; Becton Dickinson).
Stainingfor rericulocyresbearing the fraansferrin meptor. A mouse
monoclonal antihuman transferrin receptor lgGl raised against
MLA 144 Gibbon leukemic cells (Chemicon International Inc,
Temecula, CA)was diluted to IO pglmL in phosphate-buffered
saline (PBS)/l.O% bovine serum albumin. Fifty microliters of
washed cells from density gradient fractionswere suspended in 200
pL of antibody and incubated for 1 hour at 3PC. Cells were
washed three times in PBS and then incubated for 1 hour at 3PC in
200 pL of a goat antimouse IgG-FITC conjugate diluted to IO
pglmL in PBS. After washing three times, cells were stained in an
equal volume of .002% ethidium bromide for 1 hour at mom
temperature, placed on ice, and immediately analyzed by flow
cytometry.
Quanriration of protein 4.1 a and b components. RBC ghosts
were prepared from separated samples by hypotonic lysis in 40
volumes of icecold 5 mmollL Tris-HCI, 1 mmol/L EDTA, 1
mmollL diisopropylfluoro-phosphate,pH 8.0. Membranes were
solubilized and proteins were separated by sodium dodecyl sulfatepolyacrylamidegel electrophoresis (SDS-PAGE) (7% acrylamide)
according to the method of Laemmli.l* Proteins were stained with
Coomassie Brilliant Blue R-250, and the bands corresponding to
4.la and 4.lb were cut out and the dye eluted in 25% Pyridine and
quantitated spectrophotometrically.I9
From www.bloodjournal.org by guest on April 1, 2017. For personal use only.
IMPROVED ISOLATION OF RETICULOCYTES
251
Table 1. Summay of R.ticulocvte Ohhibution in High KCI and NaCI Gradients, pH 7.4
Fraction
1
2
3
4
5
6
7
8
9
10
Density(g/mL)
KCI
1.0745
67.6
(4.9)
31.E
(6.7)
1.0829
25.3
(5.2)
6.2
(1.2)
1.0880
5.2
(1.6)
1.9
(.23)
1.0919
0.3
1.0964
0.17
LO31
0.56
(.08)
1.1009
0.23
(.03)
0.27
LO71
1.1055
0.17
(.03)
0.37
(.03)
1.1102
0.17
(.03)
0.2
(0.0)
1.1148
0.17
(.03)
0.17
(.03)
1.1195
0.23
LO31
0.23
LO31
NaCl
(0.04)
1.3
(0.2)
Cellulose filtered whole blood from three subjects was separated on NaCl and KCI gradients as shown in Fig 1. Fractions correspond to the
densities indicated. Reticulocytes in each fraction were quantitated by flow cytometric analysis of ethidium bromide stained cells. Note the
significantly improved isolation of reticulocytes in the low-density fractions of the KCI gradient when compared with the NaCl gradient. Data
presented as percent reticulocytes(SEMI.
Hemoglobin A,cmea.w"nts.
The relative amount of hemoglo-
bin AI, in each gradient fraction was measured by ion exchange
high-performance liquid chromatography (DIAMAT Fully Automated Hemoglobin Analyzer System; Bio Rad, Hercules, CA).
RESULTS
We hypothesized that the CI-dependent K loss from
reticulocytes might alter their density during density gradient centrifugation, resulting in less than optimal reticulocyte separation under conditions most commonly used. In
an effort to enhance reticulocyte separation, we tested the
effect of various factors that influence the activity of
CI-dependent transport. Activation of the transport by
reduced PH,~-*'
and suppression of the transport by reducing the transmembrane Kgradient and substituting NO, for
CI were investigated?*
Efect of gradient composition on RBC and reticulocyte
distribution. Comparison of the density distribution of
total RBCs on high-K and high-Na gradients showed that
the major difference was an increase in the proportion of
cells in the two lowest density layers of the high-K gradient
(Fig 1). When the reticulocyte distributions in gradient
populations were determined, it was found that these two
least dense populations from the high-K gradient were
markedly enriched in reticulocytes (Table 1). In contrast,
reticulocytes had a much broader distribution on high-Na
gradients and did not show such remarkable enrichment in
the lowest-density populations.
If the presence of higher-density reticulocyteson high-Na
gradients were caused by CI-dependent K loss via the KCI
cotransport pathway, it should be accentuated by reducing
the pH of the NaCl medium to 6.8, the pH optimum for
activation of this pathway in cells containing HbC." As
expected, incubation and separation of the cells in high-Na
medium at pH 6.8 did result in even greater increase and
broadening of reticulocyte density (data not shown).
Effect of transient exposure to low pH in ACD anticoagulant. Having found that separation in high-Na medium at
low pH resulted in reticulocyte dehydration, we asked
whether transient exposure to low pH of blood samples
drawn into ACD anticoagulant would affect reticulocyte
distribution during subscqucnt density separation on high-K
gradients at pH 7.4. Parallel samples were drawn into ACD
(final pH 6.4 to 6.7) and EDTA and were then immediately
separated on KCI gradients at pH 7.4. A perceptible shift to
higher density was observed in the ACD sample, and the
percentages of reticulocytes in the lowest-density layers
were reduced (data not shown). When the ACD was
adjusted to pH 7.4 before adding the blood, the subsequent
density and reticulocyte distribution was identical to that of
thc EDTA sample. This indicated that it was exposure to
low pH, not the effect of an ACD component, that
influenced the density distribution. All of the data presented in this report were obtained using samples drawn
into EDTA.
A
Fig2 Reseparationofthetwo
towest-density cell popul.tiom
from a KCi density gradient,
showing that dehydration in
high-No medium is CI-dependent. Filtered RBCI were initially
separated on KCI gradients, and
the lowest density (A) and next
lowest density (B) populations
ware isolated, divided into 3
parts and incubated for 1 hour
(37°C) in KCI, pH 7.4, NaNOr pH
6.8, and NaCI, pH 6.8 media. They
wemthen recentrifugedon gradients prepared with the same
ionic composition of the incubation media, as indicated. Note
that density increase caused by
cell dehydrationoccurred only in
the NaCl medium.
Density
(g/mL)
1.0785
1.0829
1.0880
1.0919
1.0964
1.1009
1.1055
1.1 102
1.1148
K NO, C1
B
K N0,CI
Fraction
1
2
3
4
5
6
7
8
9
10
From www.bloodjournal.org by guest on April 1, 2017. For personal use only.
252
SORETTE, SHIFFER, AND CLARK
Table 2. Reticulocyte Distribution
1
A
B
13.0
7.1
2
4.4
1.05
3
4
5
6
7
8
9
3.7
2.0
6.5
4.1
28.4
10.4
64.8
33.1
89.9
71.3
88.4
87.7
99.3
97.0
Reticulocytematuration and KCl cotranspot? activity. Next,
we asked whether the level of KCl cotransport activity was
correlated with two indices of reticulocyte maturation:
RNA content and expression of the transferrin receptor. To
address this question, a reticulocyte enriched fraction from
the high-K gradient was reseparated on a NaCl gradient,
pH 6.8. The lowest, highest and an intermediate density
fraction were stained with ethidium bromide and monoclonal antitransferrin receptor IgG, and two-color-fluorescence analysis was performed by flow cytometry (Fig 3).
The lowest-density fraction was enriched in mature RBCs
(22% reticulocytes) with a small percentage (7.2%) of cells
staining positive for the transferrin receptor (Fig 3B). The
intermediate fraction contained a higher percentage of
reticulocytes, and a greater proportion of reticulocytes
bearing the transferrin receptor (Fig 3C). The cells having
the most active transport activity, isolated in the highestdensity fraction of the chloride gradient (Fig 3D) are a
nearly pure fraction of reticulocytes (96.0%). A significant
percentage (39%) stained positively for transferrin receptor. Moreover, these reticulocytes stained more intensly for
RNA and the transferrin receptor, suggesting that this
fraction is enriched in the youngest circulating reticulocytes. These results indicate that the least mature reticulocytes are those that become most dehydrated via C1dependent K loss.
Age-related markers in fractions from high K+ gradients.
In an effort to assess the relative age of the mature cell
Reticulocyte distribution in cells from one subject that were isolated
from low-density fractions of a KCI gradient, incubated in high-NaCI
medium, pH 6.8, and reseparated on NaCl gradients. Layers A and B
denote the lowest and next lowest density fractions from the original
KCI gradient separation. Layers 1 to 9 represent populations recovered
from the secondary NaCl gradients. Data presentedare percent reticulocytes quantitated by manual counts of 1,000 New Methylene Blue
stained cells from each fraction.
Chloride dependence of reticulocyte dehydration in high-Na
medium. To further verify that the improved separation of
reticulocytes on high-K gradients was caused by suppression of C1-dependent K loss, the effect of C1 substitution by
NO3 was tested (Fig 2). When low-density cells obtained
from a high-K gradient were incubated and recentrifuged
on a NaN03 gradient at pH 6.8, they returned to the same
density as those reseparated on a high-K gradient at pH 7.4.
In contrast, a parallel sample reseparated on a NaCl
gradient, pH 6.8, yielded a markedly broadened density
distribution attributable to cellular dehydration. Quantitation of reticulocytes in each of the density fractions showed
that it was the reticulocytes that became dehydrated, thus
concentrating in the most dense fractions of the NaCl
gradient (Table 2).
4
B
3
3
2
2,
I
1
,
I
1
2
3
Log Green Fluorescence
(FITC-anti-transferrin receptor)
4
Fig 3. Two-color fluorescence
analysis for RNA content and
transferrin receptorfor 3 cell populations from secondary, NaCl
density-gradient separation. After cells were separated as indicated in Fig 2, the least dense (6).
an intermediate density (C, density 1.1009 t o 1.1055 g/mL) and
the most dense (D) cell populations were stained with ethidium
bromide and FITC antitransferrin
receptor antibody. Contour plots
from flow cytometric analysis of
the three populations and an unseparated control sample (A) are
shown. Note the progressive increase in ethidium bromide staining from lowest t o highest density, and concomitant increase in
staining for the transferrin receptor. The high intensity of staining
for both markers in the most
dense fraction indicates that the
youngest reticulocytes are the
cells that become most dehydrated in NaCl medium.
From www.bloodjournal.org by guest on April 1, 2017. For personal use only.
IMPROVED ISOLATION OF RETICULOCYTES
253
I
I .07
1.08
1.09
1.10
1.11
1.12
1.11
1.12
Density (g/mL)
4
0.4
I
'
0.2
I .07
I
1.08
1.09
1.10
Density (g/mL)
Fig 4. (A) The relative proportion of hemoglobinAlc increasedwith
increasing mean cell density in high-K gradients. The low proportion
of HbA
in the highly selected reticulocyte-enriched fractions is
consistent with a young mean cell age. (n = 4, mean f SEM). (B)
Ouantitation of protein 4.la and 4.16 from the same gradient populations shows a progressive increase in the 4.la component though the
lowest-density fractions in the gradient. There was no consistent
further increase of the 4.la component in the highest-densitypopulations. (N = 4, mean & SEM).
fractions throughout the high-K gradients, we assayed two
properties that have been proposed to reflect aging of
mature RBCs: the percentage of Hb in the AI, form, and
the relative proportions of the a and b components of
protein 4.1. As has been previously shown for other density
gradient
the relative proportion of hemoglobin in the A fraction increased with increasing mean cell
density (Fig 4A). Quantitation of protein 4.la and b
performed on the same blood samples showed that there
was a significant increase in the 4.la component through
the least dense 10% of the cell distribution, but this
difference was less pronounced throughout the rest of the
density separated population. Neither HbAI, nor the 4.la
component was particularly increased in the highestdensity populations (Fig 4B).
DISCUSSION
Several density-based or rate-sedimentation techniques
for separating reticulocytes from erythrocytes, and for
separating erythrocytes into populations of differing mean
cell age, have been d e ~ e l o p e d . ~
Our
- ~ study differs from
these in showing that highly enriched populations of reticulocytes can be obtained from normal blood in a one-step
density separation on gradients containing a high-K concentration to approximate the internal cation composition of
RBC. This method results in isolated populations of up to
80% reticulocytes, a much greater enrichment of normal
reticulocytes than previously achieved using high-Na medium or other density-based methods.25 For even greater
reticulocyte enrichment, low-density populations isolated
on KCI gradients are incubated and recentrifuged in NaCl
medium, which promotes selective increase in density of the
youngest reticulocytes. The most dense fractions from the
secondary separation are almost all reticulocytes. Although
affinity based techniques also yield highly enriched reticulocyte populations, they only select fibronectin or transferrin
receptor-bearing reticulocytes, and recovery of the cells
after isolation in an undamaged state is problematic.
We have shown that the improved isolation of reticulocytes in high-K medium is attributable to the suppression of
KCl loss and cell dehydration via the K-Cl cotransporter,
which is active in reticulocytes but is suppressed upon
erythrocyte maturation.% It should be noted that even if
high-K gradients are used, reticulocyte separation is less
effective if the blood is drawn into ACD anticoagulant. This
is caused by transient activation of KCl cotransport resulting in irreversible dehydration of some reticulocytes. This
mechanism of pH effects on RBC-density-distribution
differs from that discussed by Walter et al.27 They found
that separation at low pH resulted in an increase in volume
and a decrease in the density of mature rat RBC. Although
this would cause increased overlap in density between
reticulocytes and erythrocytes, the swelling of mature cells
would be largely reversible with return to pH 7.4, whereas
the KCl cotransport mediated dehydration of reticulocytes
would not.
The distribution of putative markers for aging of mature
RBCs HbAI, and protein 4 . 1 ~was consistent with the
reticulocyte distribution, showing low levels of both markers in reticulocyte-enriched cell populations. There was a
progessive increase in both markers with increasing cell
density, but this tended to plateau in the last few most
dense populations. This suggests that although there may
be a general increase in average cell age with increasing
density of layers from the KCl gradients, the very highest
density cells may not represent a highly enriched population of the oldest cells.
These studies show a convenient, highly effective approach for selective separation of reticulocytes from mature
erythrocytes in normal blood samples via exploitation of
their C1-dependent K transport function. We anticipate
that its capacity to provide i m y m x d recovery of reticulocytes in free suspension will facilitate investigation of a wide
variety of fundamental biological questions.
ACKNOWLEDGMENT
The authors thank Margaret Rheinschmidt for her generous
consultation on the flow cytometric analysis of reticulocytes. We
also gratefully acknowledge both the technical expertise and
theoretical support from Steve Tanaka on the HbAl, analysis. We
acknowledge the technical assistance of Hung Manh Nguyen.
From www.bloodjournal.org by guest on April 1, 2017. For personal use only.
SORETTE, SHIFFER, AND CLARK
254
REFERENCES
1. Key J A Studies on erythrocytes, with special reference to
reticulum, polychromatophilia and mitochondria. Arch Intern
Med 28:511,1921
2. k i f RC, Vinograd J: The distribution of buoyant density of
human erythrocytes in bovine albumin solutions. Proc Natl Acad
Sci USA 51:520,1964
3. Corash L Density dependent red cell separation, in Beutler E
(ed): Red Cell Metabolism. New York, NY, Churchill Livingstone,
1986, p 90
4. Danon D, Marikovsky Y Determination of density distribution of red cell population. J Lab Clin Med 64:668,1964
5. Turner BM, Fisher RA, Harris H: The age related loss of
activity of four enzymes in the human erythrocyte. Clin Chim Acta
5085,1974
6. Desimone J, Kleve L, Shaeffer J: Isolation of a reticulocyterich fraction from normal human blood on renografin gradients. J
Lab Clin Med 84:517,1974
7. Rennie CM, Thompson S, Parker AC, Maddy A Human
erythrocyte fractionation in “percoll” density gradients. Clin Chim
Acta 98:119,1979
8. Vettore L, De Matteis CM, Zampini P: A new density
gradient system for the separation of human red blood cells. Am J
Hematol8:291,1980
9. Fabry ME, Mears JG, Pate1 P, Schaefer-Rego K, Carmichael
LD, Martinez G, Nagel R L Dense cells in sickle cell anemia: The
effects of gene interaction. Blood 64:1042,1984
10. Tannert C, Tsamaloukas A Enrichment of reticulocytes by
lectin mediated agglutination. Acta Biol Med Germ 40:969,1981
11. Light ND, Tanner MA: An affinity chromatography method
for the preparation of human reticulocytes. Anal Biochem 87:263,
1978
12. Brun A, Gaudernack G, Sandberg S: A new method for
isolation of reticulocytes: Positive selection of human reticulocytes
by immunomagnetic separation. Blood 76:2397,1990
13. Beutler E, West C, Blume KG: The removal of leukocytes
and platelets from whole blood. J Lab Clin Med 88:328,1976
14. Beutler E, Gelbart T The mechanism of removal of leukocytes by cellulose columns. Blood Cells 12:57,1986
15. Herrick I, Adams MF, Huffaker EM: Chem Abstr 67:
91878q, 1967
16. Brecher G: New Methylene Blue as a reticulocyte stain. Am
J Clin Pathol 19:895,1949
17. Sage HB, O’Connell JP, Merolino TJ: A rapid, vital staining
procedure for flow cytometric analysis of human reticulocytes.
Cytometry 4:222,1983
18. Laemmli U K Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680,1970
19. Fenner C, Trout RR, Mason DT, Wickman N, Coffelt J:
Quantitation of Coomassie Blue stained proteins in polyacrylamide gels based on analysis of the eluted dye. Anal Biochem 63:595,
1975
20. Ellory JC, Hall AC, Ody S A Factors affecting the activation
and inactivation of KCl cotransport in ‘young’ human red cells.
Biomed Biochim Acta 49:S64,1990
21. Brugnara C, Kopin AS, Bunn HF, Tosteson DC: Regulation
of cation content and cell volume in hemoglobin erythrocytes from
patients with homozygous hemoglobin C disease. J Clin Invest
75:1608,1985
22. Canessa M, Fabry ME, Blumenfeld N, Nagel R L Volume
stimulated, C1-dependent K+ efflux is highly expressed in young
human red cells containing normal hemoglobin or HbS. J Membr
Biol97:97,1987
23. Boyd EM, Thomas DR, Horton TF, Huisman THJ: The
quantities of various minor hemoglobin components in old and
young human red blood cells. Clin Chim Acta 16:333,1967
24. Nakashima K, Nishizaki 0, Andoh Y, Takei H, Ita1 A,
Yoshida Y Glycated hemoglobin in fractionated erythrocytes. Clin
Chem 35:958,1989
25. Rushing D, Vengelen-Tyler V Evaluation and comparison
of four reticulocyte enrichment procedures. Transfusion 2786,
1987
26. Ellory JC, Hall AC: Evidence for the presence of volumesensitive KC1 transport in ‘young’ human red cells. Biochim
Biophys Acta 858:317,1986
27. Walter H, Krob EJ, Wenby RB, Meiselman HJ: Differential
effect of pH on the density and volume of rat reticulocytes and
erythrocytes. Relevance to their fractionation by centrifugation.
Cell Biophys 16:139,1990
From www.bloodjournal.org by guest on April 1, 2017. For personal use only.
1992 80: 249-254
Improved isolation of normal human reticulocytes via exploitation of
chloride-dependent potassium transport
MP Sorette, K Shiffer and MR Clark
Updated information and services can be found at:
http://www.bloodjournal.org/content/80/1/249.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.