Download Modified Red Blood Cells

From www.bloodjournal.org by guest on August 10, 2017. For personal use only.
Osmotic
Hemolysis
Modified
Red
By Ricii
T
HE
MECHANISM
for
on
the
for
Kochen5
hemoglobin,
break
using
emerging
the
examined
shadow
a gross
cast
that
opened
a circular
ghosts
It
holes
and closed
zone about
It
served
was
by exposure
to warrant
was
finger
red
apparent,
report
which
puncture
Angeles,
0.15
or from
California)
M
sodium
cells
in
chloride
were
the
by
cell
cell
many
a subject
centered
by
way
small
in the
gradual
and
Seeman7
of
of
a
openings.
opposite
direction.
decrease
red cells,
rapid
however,
the
order
in salt
and
also
osmotic
using
of
The
that
the
200
holes
its origin
in an attempt
of dialysis
against
a
however,
shock.
electron
AU
in
were
mi-
diameter
localized
to arrest
fixative
in
hemolytic
properties
appeared
diffusion
resulted
in
hemolysis
hypotonic
phenomenon
had
to be
been
enough
obmodified
interest
follows.
used
Hyland
ACD-plasma.
before
of
been
has
the
effect
a gradual
of hemolyzing
by
membrane
here
had
the
device
soon
blood
of
way
has
was characterized
by hemoglobin
while
rapid
hemolysis
probably
MATERIALS
Human
out
of the
the reaction
of a red
cell whose
to the fixative.
The phenomenon
the
discussion
during
hypotonic
hemolysis.
one micron
in diameter.
The work
reported
various
stages
by
solution.
cells
the
by
resin
to
pictures
shown
the
of red
of
a dye which
formed
a precipitate
with
to be a single
stream
of hemoglobin
produced
was
in
or
as a recoil
that slow hemolysis
surface
of the cell,
R. Gruis
NAoMI
diffuses
and
appeared
as well
Cells
Much
membrane,
ion exchange
contrast
cine
rupture.
croscopy,
at
cell
Blood
hemolysis
hemoglobin
the
Chemically
AND
years.
microscopy
what
using
an
made
phase
It was concluded
over
the entire
in
in
light
observed
from
Danon6
content,
many
of whether
large
BiiR
of osmotic
discussion
question
single
F.
of
use,
AND
throughout
Blood
Bank
The
cells
except
METhoDs
this
when
study.
(Hyland
were
the
They
Division,
washed
effect
three
of multiple
were
obtained
Travenol
times
Labs.
in
washes
either
by
Inc.,
Los
10 volumes
was
of
studied.
Clutaraldehyde
(25 per cent stock,
Fisher
Scientific
Company)
was distilled
before
use,
and diluted
with either
distilled
water
or with 0.15 M sodium
chloride.
Osmolarities
were
measured
on a freezing
point osmometer
(Osmette
Precision
Systems
Company,
Framingham,
Massachusetts).
From
the Department
of Microbiology,
School
of Medicine,
University
of Southern
California,
Los Angeles,
California.
This work
was supported
by Grant
HE-07976
from
the National
Heart
Institute,
U.S.
Public Health
Service.
First submitted
April 2, 1968; accepted
for publication
August
5, 1968.
RICHARD
F. BAKER,
PH.D.:
Professor
of Microbiology,
School
of Medicine,
University
of Southern
California,
Los Angeles,
California,
NAoMI
R. CILLIS:
Research
Associate,
Department
of Microbiology,
School
of Medicine,
University
of Southern
Calif ornia,
Los
Angeles,
California.
170
From www.bloodjournal.org by guest on August 10, 2017. For personal use only.
OSMOTIC
Fig.
of red
1.-Slide-coverslip
preparation
cells after six minutes
dialysis
0.2 per cent
glutaraldehyde
in
water.
Five of the cells show
against
distilled
well
developed
is
171
HEMOLYSIS
on
seen
detached
optics-
crowns;
cell
crown
a small
at lower
at upper
crown
right,
right.
and a
Phase
X 700
Dialysis
Dialysis
in
a
carried
was
cellophane
bag
was
immersed
experiments
and
1 per
cent.
time
from
the
microscopy
5
ml.
of
of
osmium
and
red
in
cell
were
and
distille(1
population
made
water
at
as
which
\V
for
a
saline
The
function
of
examined
were
thin
isotonic
California).
temperature.
between
.05 per
were
aliquots
of
In
cent
room
varied
withdrawn
Vestopal
in
1 ml.
Angeles,
preparations
of larger
embedded
Los
was
were
coverslip
in
cells
Inc.,
concentration
Slide
Pellets
packed
Rogers,
glutaraldehyde
dialysis.
tetroxide
ml
.05
Waters
ghttaraldehyde
Samples
start
suspending
(Van
the
(Reichert).
cent
by
bag
in
indivi(lIIal
per
out
dialysis
elapsed
by
postlixed
sectioning
phase
with
and
1
electron
microscopy.
Two-Step
Method
.05
in
ml.
packed
isotonic
a function
as
shocked
and
at 11,500
and lower
The
more
eleCtrOn
microscopy
Age
designed
for
small
which
had
saline
moment
of
was
of
fixed
done
blood
been
in
as
these
water.
with
1 ml.
of glutaraldehyde
Samples
cells
then
After
an excess
described
were
withdrawn
were
osmotically
1 minute
of 5 per
of
exposure
cent
glutaraldehyde.
of the
determinations.
al)ove.
was
less
variable.
and
distilled
samples8
ACD
with
mixed
was
mixing,
5 volumes
effectively
were
by
than
used
6 days
for
most
were
centrifuged
for
5 minutes
rpm.
in a microhematocrit
10 per cent of the column
elaborate
and
precise
in a linear
two
isotomc
concentration
to
were
of Cells
cells
segregation
The
them
the cells
red
of
the
from
introducing
A method
1 ml.
iii
glutaraldehyde
water
Separation
\Vashed
The
of time
by
to distilled
Phase
cells
saline.
fractions
at
density
either
centrifuge
(Clay-Adams)
following
which
the upper
were isolated,
corresponding
to young
and old fractions.
method
of separation
according
to buoyant
density
gradient
of bovine
serum
albuniin9
was used on one occasion.#{176}
en(l
of the column
were
taken
as representative
of young
and
01(1 cells.
RESULTS
With
taken
the
at one
after
4-6
cells,
ghosts,
glutaraldehyde
minute
minutes
and
concentration
intervals
of dialysis
partially
cells
showed
a precipitated
contact
with
it over
an area
For
the
sake
of convenience
All stages
of crown
development
and
the
hemolyzed
fixed
examined
sample
at 0.5
by
consisted
cells.
Many
per
phase
cent
samples
microscopy.
of
3 classes
of
the
partially
were
Typically
of
cells:
intact
hemolyzed
mass
of hemoglobin
exterior
to the
cell but
in
approximately
one micron
in diameter
(Fig.
1).
we have
termed
these
protrusions
“crowns.”
are seen in a typical
preparation,
from a slight
#{176}The
authors wish to thank Dr. R. C. Leif for assistance
with this procedure.
From www.bloodjournal.org by guest on August 10, 2017. For personal use only.
172
BAKER
Fig.
2.-Section
way
of a single
and
lowest
through
at
on itself.
x
red
membrane
the
opposite
23,400
Insert
cell
break.
showing
characteristic
Hemoglobin
pole
of the
(corner)
Note
the
the
by
at the
1)reak
membrane
curled
back
at higher
magnification.
is highest
broken
membrane
loop
(;ILLIS
of hemolysis
concentration
cell.
shows
features
AN!)
x 40,000
dimple
from
in
the
An
Figure
break,
The
the
cell.
cell
surface
Detached
electron
to
crowns
micrograph
broken
membrane
Stages
in
takes
place
an
area
of
hemoglobin
through
section
curled
around
on
nearly
a typical
high
concentration
concentration
at the
is seen
are
a few
seen
in
Figures
hundred
Angstrom
detached
is seen
cell
of hemoglobin
opposite
side
itself
on
one
3a,
cell
(Fig.
hemoglobin
3d)
and
the
remaining
membrane
in
appears
the
ghost
nearly
is
clumped,
b,
c, d.
Initial
units
in
diameter
intact.
and
The
is
of
the
rupture
(Fig.
is
small
seen
the
cell.
microscopy
membrane.
3a).
The
diameter
of the maximum
area
of membrane
disruption
order
of one micron.
In the late stages
the crown
is barely
attached
of
in
near
of the
side
the impression
gained
from
phase
circular
area
is torn
out of the cell
development
over
seen.
the
low
sections
support
approximately
crown
mass
often
of a thin
2, which
illustrates
with
a corresponding
crown.
Serial
that
a single,
a large
are
of
to
the
the
amount
by
phase
From www.bloodjournal.org by guest on August 10, 2017. For personal use only.
OSMOTIC
173
HEMOLYSIS
Fig.
3.-(A)
Earliest
stage
of membrane
disruption
in red cell after
glutaraldehyde
treatment
followed
by osmotic
shock.
A membrane
component
is lifting
off
in the manner
of a flap.
A homogeneous
material
lies between
the flap and
the cell
surface.
X 56,500.
(B) Hemoglobin
has started
to emerge
through
an opening
0.5.
in diameter.
X
thin
section
in diameter.
32,700.
attached
to the
Note
(arrow)
and
(D)
ghost.
the
Large
(C)
both
inside
X 11,450.
The
loops
precipitated
membrane
of
mass
outside
the red cell.
Late
stage
of hemolvsis.
is nearly
membrane
inside
the
granularity.
by high
power
of hemoglobin
The
is seen
in this
membrane
break
is about
2j
The
hemoglobin
mass
is barely
intact
in the
plane
of this
ghost,
in
vicinity
of
the
section.
the
break.
in
ghosts
electron
17,800.
x
microscopy
as an intercellular
which
lacked
crowns,
either
microscopy
Two
of
Step
Results
obtained
of fixation
time
which
4 as t,.
producing
One
washed
with
cells
the
could
be
production
red
cells
to the
of cells
fixation.
in shifting
change
number
Variations
the curve
in shape.
of
in saline,
by dialysis.
Using
of fixation,
and salt
Figure
showing
of crowns
in
4 to
fixation
in Figure
hemolyze
decreases
glutaraldehyde
Figure
to the
The
is shown
of the cells
of crowns
this
con-
4 relates
crowns
of glutaraldehyde.
of fixation
less than tmax many
for longer
times
the number
prolonged
glutaraldehyde
independently.
cent
concentration
maximum
to
obtained
time
varied
in per
a specified
corresponds
resulted
without
exposing
were
similar
to those
of fixative
concentration,
the
For times
a crown;
with
Repeated
first
of crown
time
scale
by
around
efficiency
merely
time
or
sections.
osmotic
shock,
the variables
centration
zero
seen
?%Iethod
then
to
method
the
serial
Holes
were
never
phase
microscopy
without
toward
concentration
the
right
in
ACD)
or
left
on
the
Washings
volume
with
of
l)lood
10 volumes
from
of 0.15
ACD
storage
M sodium
(6
chloride
days
repeatedly
at
4 C.
at room
was
tern-
From www.bloodjournal.org by guest on August 10, 2017. For personal use only.
174
BAKER
AND
GILLIS
U)
0.2
z
%
0
C)
I
IU)
-J
-I
It
mox
w
C)
2
3
4
TIME
Fig.
minutes
4.-Percentage
of fixation
(MIN.)
of red cells
in 0.2 per cent
5
OF
6
FIXATION
showing
crowns
glutaraldehyde.
plotted
as a function
of time
The fixation
time
corresponding
to the maximum
of the curve
is denoted
as trnax. The efficiency
of crown
is low on the left side of the curve
because
ghosts
are being
formed;
right side because
fixation
has progressed
too far to allow hemolysis.
perature,
and
creased
linearly
cells
did
a determination
with
consistently
old
tmax
number
reached
made
was
after
of washings
a peak
of crown
as
each
shown
washing.
in
production
at
production
low on the
tlnax
Figure
in
5. Young
shorter
times
than
cells.
Localization
The
washed
of
the
in
of Crown
optimum
red cells
a glass slide
After
rapid
concentration
(ACD
was made
air drying
for 20 seconds.
tion was
restricted
Phase
slide
No
(Fig.
cell. Control
of the glass
side of the
orientation
Sites
6).
was
fixation
time
determined.
of cells after
such optimal
the slide
was immersed
examination
the rim
to
crowns
experiments
throughout
of
were
disks
cells
relative
in glutaraldehyde
A conventional
fixation
vertically
seen
attached
lying
parallel
to the glass
to
that the
subsequent
the
that
crown
to the plane
biconcave
cells
for
smear
unon
for crown
production.
into
distilled
water
of the wet slide
showed
the cell, that
is, parallel
(Fig.
7) showed
the fixation
and
biconcave
of the
and
storage)
region
adhered
of
the
the surface
with
the flat
to
hemolysis,
to the plane
of the
was fixed at all times.
formaof the
slide.
Thus
the
DIscussIoN
The first question
lysis described
here
or whether,
in
fact,
to be discussed
represents
the
the
development
is as to whether
the
mechanism
of normal
of hemoglobin
crowns
single
break
hemoosmotic
hemolysis,
is the
reaction
to
From www.bloodjournal.org by guest on August 10, 2017. For personal use only.
OSMOTIC
175
HEMOLYSIS
ISI-.
‘4
12
z
Io
C.)
8
x
4
EFFECT
OF MULTIPLE
i
PIG OF
Fig.
5.-The
shown
for
here
effect
for
maximum
mirn
number
than
for
young
of
Several
fixative.
110 AU,
hypotonic
WASHINGS
multiple
IN ISOTONIC
SALINE
in isotonic
saline
washes
a red
one.
It
if present
hemolysis.
cell
whose
lines
has
in
properties
of evidence
been
have
indicate
been
that
established7’10
that
and
hypotonicity,
However,
cedure;
that
mechanical
crown
production
the hemolyzing
solution,
Particles
of colloidal
gold
or by
a prior
crowns
is,
osmotic
properties
treatment
have
shock
of
with
been
not
followed
the
modified
the
is
time
maxitimes
than
large
fixative
by
is
with
a
of
with
It
been
diameter
cell
AU
during
are ex-
250 AU would
not
as one micron.
It is
application
of fixative
followed
fixative.
have
contact
explanation
will
enter
a red
larger
than
250
demonstrable
membrane
by
latter
ferritin
eluded.
The
exclusion
of colloidal
material
larger
be consonant
with
the existence
of an opening
as
relatively
easy
to produce
crowns
by a simultaneous
shock.
on
cells (Y) and old cells (0). The fixation
increases
with repeated
washings.
The
of old cells
is reached
at longer
fixation
cells.
with
correct
.;p
a population
of young
crown
production
(trnax)
of crowns
in the case
hypotonicity
the
of
WASHES
by
the
seems
altered
hypotonic
reverse
clear
by
prothat
the
the
fixative.
It has been
shown
by Seeman7
that
pores
in the membrane
about
200 AU
in diameter
open
and close during
hypotonic
hemolysis.
The ghost
so produced
is impermeable
to hemoglobin
and
may
regain
its former
selectivity
to ion
transport.1114
Clearly
the membrane,
although
breached
at many
sites,
has
repaired
itself.
The
evidence
for membrane
repair
in the case
of
the much
larger
openings
Ghosts
phase
microscopy,
are
with
seen.
In
produced
large
thin
and
little
or no
through
the edge
holes
but
sections
by
in
they
a
crown
wet
are
the
extrusion
may
would
although
preparation
never
seen
appearance
loss in membrane
of a large
opening.
of
a ghost
continuity
could
However,
serial
be
summarized
be
as follows.
easily
detectable
many
detached
with
be
an
with
crowns
attached
interpreted
sections
through
crown
as
a cut
such
a
From www.bloodjournal.org by guest on August 10, 2017. For personal use only.
176
BAKER
AND
CILLIS
Fig.
6.-Red
cells
treated
with
0.25
per
cent
glutaraldehyde
were
spread
on a glass slide by a conventional smear technique.
Osmotic
shock
the
of
spread
cells
produced
crowns
emerged
in a direction
parallel
to the slide,
i.e., localized
on the rini
of the cell. Some
detached
crowns
are
seen.
Densities
are
reversed
in
this
figure
and crowns
appear
black.
Phase
optics
x 700.
which
Fig.
7.-Control
after
0.25
per
hypotonic
hemolysis.
x 400.
hemolysis.
cell
as
well
support
membrane
the
It
has
fixative
view
repair
been
for
fixation.
with
known
by
that
protein
dehyde.
the result
hemoglobin
as
since
the
may
it is likely
constituent
membrane
insofar
repair
as repair
could
take
as
is an
Polymerization
of thorough
with
low
with
efficient
formation
not
it
the
lipid
cross
linking
of
slip
after
sections
tend
to
that
is,
as
a
tissue
preserved
by
apparent
if
that
lipids
the
are
dehydration
and
is a most
important
it therefore
seems
mild glutaraldehyde
agent
be
slide
and
opening,
well
were
appears
is concerned,’618
place
even after
Thus
it might
will be denatured
fixation.
Short
concentrations
cell
by
the
cover
introduced’5
extracted
that
with
a large
are
membranes
Thus,
be
Since
imino
groups.
hemoglobin
random
were
However,
tetroxide.
of cells
to glass
seconds,
air drying
field
with
membranes
procedures.
and
well
left
aldehydes
and
Same
of
not
that
adherence
for 30
(B)
hundreds
are
the
alone.
in osmium
fixation
400
x
of
microscopy
Glutaraldehyde
amino
smear.
examination
fixation
postfixed
embedding
Dry
demonstrating
fixation
that
the ghosts
has occurred.
electron
stabilized
membrane
plausible
(A)
as the
glutaraldehyde
tissue
were
not
red cell smears
glutaraldehyde-saline
cent
for
proteins,
reacting
expected
that
membrane
and stabilized
by glutaral-
aggregates
periods
of fixation
of glutaraldehyde
of
hemoglobin
will
be
of highly
concentrated
might
result
in poly-
From www.bloodjournal.org by guest on August 10, 2017. For personal use only.
OSMOTIC
177
HEMOLYSIS
mers
of
hemoglobin
(200
AU
). With
via
the
normal
result.
On
the pressure
that
too
If the
the
large
internal
small
pores
Heterogeneity
Murphy2’
lesterol
a large
it is possible
by a large
in chemical
in protein
lary
exists
cells
must
composition
also
a difference
area,
times
not
over
as
an
the
diffusion
would
never
open,
be
it would
surface
demonstrable,
cell
of
a result
of
the
and
logical
cell.
than
in
coincidence
that
by
young
young
cells
area
is known
washes
in
isotonic
a corol-
the
fact
that
cells
for
maximum
and
but
with
That
documented
cells.22’23
Another
young
the
little
fragility.
than
until
that
chothat
differ-
properties
multiple
between
abundant
Too
i5 shown
tmax
times
behavior
are
surface.
osmotic
and
for longer
however,
cholesterol,
of course
visco-elastic
of
increasing
fragility
to fixative
forms24
more
then
the
on
diameter
and
membrane
pores
with
tritiated
rim. It is possible
correlation
reflect
in visco-elastic
stromalytic
in old cells.25
normal
entry
in the
small
(Fig.
5). It has been
abundantly
more
fragile
than
young
red
that
be
was
exist
useful
osmotic
exposed
crown
production
cells are osmotically
It may
may
between
be
the
over its
anywhere
constitution
to permit
any
membrane
composition.
The
shift
of tflax
to longer
here
of the
to water
break
that
break.
autoradiography
arotmd
the
time
reported
pores
due
crowns
appear
exclusively
on the rim of the cell
in the visco-elastic
properties.
There
is evidence
factors
of the red cell reside
in the membrane.19’2#{176}
showed
by
was concentrated
at this
of
possible,
membrane
were
homogeneous
break
would
occur
at random
is not
the case-the
pointing
to a difference
that
shape
determining
saline
through
increasing
not
the other
hand,
must be relieved
This
ences
to diffuse
pressure
that
old
of membrane
old red
indication
red
almost
old
cells
is
non-existent
disruption
for
crown
formation
is about
the same
in shape
and size as the area of hemoglobin
extrusion
observed
by Kochen5
and
that
inferred
by Seeman7
from
electron
micrographs
of thin
sections.
It has
been
shown
here
that
the
membrane
break
for crown
formation
occurs
only on the rim of the cell. The
site of the
membrane
pores
reported
finding
if they were
shown
by Seeman
to be localized
is unknown;
also to the
it would
rim of the
be a significant
cell.
SUMMARY
The
mechanism
investigated
hemoglobin
as
large
the
cells
osmotic
mild
(crown)
as
2/h
believed
that
Hemoglobin
of
of
after
red
is
in
repair
extrusion
require
Both
longer
A correlation
seen
diameter.
membrane
cell.
hemolysis
fixation
old
in
around
a
Ghosts
with
takes
by
this
and
young
than
fixation
with
previous
del
hemolyse
of
human
red
blood
A
mass
break
glutaraldehyde.
single
membrane
holes
large
are
cells
has
been
of precipitated
which
may
not
seen
and
be
it
is
place.
mechanism
red
do young
work
takes
cells
place
exhibit
only
crown
around
formation,
the
rim
but
old
cells.
on mode
of osmotic
hemolysis
of red
cells
is discussed.
SUMMARIO
Le
leve
mechanismo
fixation
in
glutaraldehyda.
IN
osmotic
Un
massa
INTERLINGUA
de
erythrocytos
human
de
hemoglobina
precipitate
esseva
es
investigate
vidite
circum
post
un
From www.bloodjournal.org by guest on August 10, 2017. For personal use only.
178
sot
BAKER
fissura
membranal
que
pote
attinger
un
diametro
de
foraturas non es vidite, e ilpare que reparo membranal
Extrusion
de
sed
hemoglobina
per
Le formation
erythrocytos.
erythrocytos
vetule
iste
coronari
require
un
plus
in
longe
Es commentate
relationes
del presente
de hemolyse
osmotic
in erythrocytos.
ad
2
GILLIS
con grande
Chimeras
es in progresso.
mechanismo
occurre
usque
AND
occurre
solo
erythrocytos
fixation
circum
tanto
que
cellulas
constatationes
con
le
vetule
como
margine
del
etiam
juvene,
juvene.
previe
studios
relative
al modos
sugar
transport.
REFERENCES
1. Ponder,
Pheuomena.
1948.
2.
E. :
New
Grune
and
Danielli,
H.,
of
University
3.
York,
and
Related
and
Stratton,
cyte
Natural
Press,
Fricke,
J. F.
Membranes.
Per-
:
Cambridge
1952.
H.:
Rate
of
escape
of
hemo-
globin
from
hemolyzed
red corpuscles.
J.
Cen. Physiol. 18:103, 1934.
4. Katchaisky,
A., Kedem,
0.,
Klibansky,
C., and DeVries,
A.: In: Copley, A. L. and
Stainsby, G., (Eds.)
Flow
properties
of
Blood. Pergamon
Press, 1960.
5. Kochen,
J. A.: Structural
disturbances
during
red cell lysis.
Amer.
J. Dis. Child.
104:537,
1962.
6. Danon,
D.:
Osmotic
hemolysis
by a
gradual
decrease
in the ionic
strength
of
the surrounding
medium.
J. Cell.
Comp.
Physiol. 57:111, 1961.
7. Seeman, P.: Transient holes in the erythrocyte
membrane
during
hypotonic
hemolysis
and
after
lysis
Cell.
Biol.
stable
by
holes
in
saponin
32:55,
and
the
1967.
of buoyant
cytes
in
bovine
Nat.
Acad.
Sci.
10.
Baker,
human
sis.
red
Nature
11.
Stein,
density
of human
albumin
U.S.A.
R.
cells
215,
Entry
during
424,
W.
solutions.
51:520,
F.:
D.:
erythrocyte
erythroProc.
1964.
of ferritin into
hypotonic
hemoly-
The
permeability
of
Cell. Res. 11:232,
ghosts.
Exp.
1956.
12. Hoffman,
J. F.: Physiological
characteristics
of human
red blood
cell ghosts.
J.
Gen. Physiol.,
42:9,
1958.
13. Teorell,
T.: Permeability
properties
of
erythrocyte
ghosts.
J. Cen. Physiol.
35:699,
1952.
14.
LeFevre,
P. C.:
K., and Barrof cellular
ultrastructure
and
enzymatic
activity
by
aldehyde
fixation.
J. Cell. Biol. 17: 19, 1963.
16. Kavanau,
J. L. : Structure
and Function in Biological
Membranes,
Vol. 2, San
Francisco,
Holden-Day,
Inc.
1963,
p. 411.
17.
Dervichian,
D. C. : In: Danielli,
J. F.,
and Pankhurst,
K. A., and Riddiford,
A. C.
( Eds. ) Surface
Phenomena
in Chemistry
and
Biology. London,
Pergamon
Press, 1958,
p.
Persistence
in
erythro-
:
70.
18.
Bangham,
A. D.:
In:
Derenck,
A. V.
S.
and Knight,
J., (Eds.)
Cellular
Injury,
Ciba
Found.
Sympos.
Boston, Little Brown
and
Co., 1964.
19. Kates,
M., Allison,
A. C. and James,
T.:
Phospatides
their
role
in
phys.
Acta
48:571,
S.:
of
Nakao,
human
blood
spherocytosis.
cells
and
Biochem.
Bio-
1961.
M.,
Nakao,
T.
and
Yamazoe,
Adenosine
triphosphate
and
shape
of
erythrocytes.
J. Biochem.
49:487,
1961.
21.
Murphy,
J. R.: Erythrocyte
metabolism
VI. Cell shape and
the location
of
cholesterol
Lab.
in
22.
certain
J.
membrane.
J.
age.
Cell.
Comp.
of
their
Physiol.
1958.
23.
P. A. and Johnson,
Marks,
lationship
for
erythrocyte
Med.
physiological
51:415,
the
65:756,
1965.
Hoffman,
J. F.: On the relationship
erythrocyte
characteristics
to
Clin.
rocytes
1967.
mediated
1961.
Sabatini,
D. D., Bensch,
J.
The preservation
R.
20.
J.
lysolecithin.
of
191:970,
nett,
membrane
8. Herz, F., and Kaplan,
E.: A microtechmc for the separation
of erythrocytes
in accordance
with their
density.
Amer.
J. Clin.
Path. 43:181,
1965.
9. Leif,
R., and Vinograd,
J.: The distribution
ghosts
Nature
15.
Davson,
meability
Hemolysis
between
and
the
their
osmotic
separating
J. Clin.
24.
stromalytic
molysis
11:494,
25.
Structural
R.
F.:
forms
of
red
blood
1964.
Mankovsky,
A. B.:
human
The
fine
produced
cells.
Y.,
differences
by
J.
stained
A basis
erythrocytes.
structure
osmotic
Ultrastruc.
and
red
of
heRe.s.
Danon,
between
Re-
eryth-
resistance:
and
37:1542,
Baker,
of
old
1958.
young
Invest.
age
old
young
negatively
branes.
J. Ultrastruc. Res. 20:83, 1967.
cell
D.:
and
mem-
From www.bloodjournal.org by guest on August 10, 2017. For personal use only.
1969 33: 170-178
Osmotic Hemolysis of Chemically Modified Red Blood Cells
RICHARD F. BAKER and NAOMI R. GILLIS
Updated information and services can be found at:
http://www.bloodjournal.org/content/33/2/170.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.