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1948 3: 465-469
A STUDY OF SICKLING OF YOUNG ERYTHROCYTES IN SICKLE CELL
ANEMIA
JANET WATSON
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A STUDY
OF
SICKLING
OF YOUNG
ERYTHROCYTES
CELL
ANEMIA
By
T
HE COMPARATIVE
tients
with
active
comment
by several
and
Shapiro3
to
part
to the
students
was
tendency
of a crisis
disease.”
of the
2,
This
3
red
cells
might
on aging,
The
.
in the plain
be due
in which
fact
smear
in pato arouse
led Murphy
crises
to sickle
be beneficial
cells
normoblasts
striking
observation
of hemolytic
of itself
of sickle
seemed
to lend further
of sickle
cell patients,
in the sickled
form.
Ia,
occurrence
might
percentage
therefore
of sickled
reticulocytes
and
disease
has been sufficiently
the
SICKLE
M.D.
WATSON,
of that
that
increasing
a higher
wards
smears
ulocyte
scarcity
sickle
cell
postulate
the reticulocytosis
showed
JANET
IN
that
before
their
crises
in
case
patient
than
after-
support
to this theory.
In reviewing
many
blood
I was able to find only two normoblasts
and one reticA quantitative
study
of the sickling
of reticulocytes
undertaken.
MATERIAL
Three
Kings
patients
with
County
active
sickle
were
chosen
Hospital
high percentage
ate mixture
was
cell
ofreticulocytes
and sickle
cells in their
used as an anticoagulant
for the venous
Oxalated
solution.
was
normoblasts
present.
It was
lum.
normoblasts
be counted
of the
to uneven
AND
preparation.
of the
presumably
through
37C.,
the
Hematology
Clinic
they
consistently
showed
blood.
blood
The Wintrobe
obtained.
brilliant
cresyl
parts
and
to be the
method,
This
irregularity
to sickle.
The
lowering
oxygen
mentioned
an
accelerating
tension
intervals.
sickled
by
of the
and
on
by metabolism.
full
sickling
Platelet
used
Cresyl
One
in a sealed
blue
stains
tinguished
from
and
of
the
hemoglobin
role
the
the
were
From
orthochromatic
of
blue
and
it happens
to
of the
normoblasts
in their
cytoplasm,
their
in sickling
ghosts
has
obtained
were
mature
could
in
different
largely
of the
distribution
ability
due
red
may
been
in the
presented
by lysis
of their
of Medicine,
Long
recently
College
state.
Two
blood
also
be
found
incubation
of
the
Further
who
their
of Medicine,
Brooklyn,
that
to sickle.
N. Y.
not
that
none
at 37
C.,
have
showed
layer
that
is easily
latter
evidence
ability
bully
.
of normoklts.
but
normoblasts
by Ponder,
lost
The
network
It was
surprising.
they
B.).
sickling
reticular
2.4 hours
orthochromatic
is not
465
after
sickled
the
hemoglobin,
Island
(W.
the
normoblasts,
The
normoblasts.
sickled
to sickle
patient
to study
reticulated.
basophilic
Since
one
in order
polychromatophilic
be
were
to sickle.
from
above,
basophilic
normoblasts
failed
Department
the
staining
however,
into
was
as described
unless
bodies,
made
the
cytoplasm
diffuse
marrow
made
polychromatophilic
of hemoglobin
cytes
of sternal
normoblasts
basophilic
Howelljolly
cc.
preparation,
orthochromatic
most
cases
of reticu-
reticulocytes
is probably
the
all
occasional
of sickling
others,
effect
cent
This
as the
packed
irregularity
per
In
as the
as soon
were
sickling
the
in o.8
preparation.
as well
which
progressive
been
have
at frequent
reticulocytes
of
blue
slip
reticulocytes
reticulocytes
because
has
which
slide-cover
sickling
the
of the
immature
however,
leukocytes,
for
included
most
most
last
this
per cent
the standard
examined
This
that
of the
ofo.
..
( b) Bone Marrow.
the
a
oxal-
afactor.6
was
of
RESULTS
to make
hours.
impression
appeared
distribution
cells,4’
the
by
equal
used
to four
of some
accurately
same
at
two
the exception
with
mixturewas
incubated
within
with
was mixed
of the
complete
red cells,
parts
drop
paraffin,
was
The
blood
One
sealedwith
sickling
not
because
Preparation
( a) Blood.
NaCI
from
study
METHODS
Sealed
I .
anemia
for
type
when
of the
whereas
containing
a full
for
of
dis-
the
quota
primary
menisco-
of
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
466
a.
SICKLED
Gas
Chamber
of
paraffin
nitrogen
the
hanging
the
was essentially
of glass.
can
The
be studied
dynamic
to sickle
cells.
Rarely
would
being
made
lost from view.
Occasionally
this method,
as well as that
3 . Gas
A
Test
test
Tube
CELL
ANEMIA
x 40
i
through
can
oil
the
quantitative
of the
permit
was
lens,
easily.
this
Here
rapidity
timing
hemoglobin
slower
mixture
immersion
great
except
of the
and
the
and
the
red cells
method
has
the
advantage
most
of the
two
minutes
two
types
of the
of sickling
result
wreath”
Because
in
reticulocytes
sickling-about
periodic
was
dioxide
Since
transformation
Sherman
‘holly
unsatisfactory.
the chamber
carbon
used.
again
of the
in the process
sickling
that
necessary.#{176} Both
blood
be watched
but
Gillespie9
found
in the
of
reticulum
forms of sickle
cells4
of these disadvantages
removal
of cells
for
counting
follows.
BLE
Patient
with
mm.,
a rubber
TA
the
blue
the formation
of the
of the sealed preparation,
would
as
with
and
was
Method
tube,
needles
which
devised
cresyl
the
impossible
outlet
saline
red cells,
the rearrangement
method
was
same
other
sickling-made
purposes
SICKLE
by Hahn
sealed
of sickling
as the
for complete
a chamber
described
directly
process
as fast
that
A water
for sickling.
drop
active
seemed
used
instead
used
were
that
IN
Method
The apparatus
made
ERYTHROCYTES
a capacity
stopper.
The
-Progressive
I
Time
of
outflow
Sickling
cc. was
(per
upon
Sickle
100 RISC)
(per
I
a gas inflow
with
of Reticuloc.ytes
Reliculocytes
in (‘1)2
set up with
equipped
was
a water
Aeration
Cells
100 RBC)
and
seal.
with
outflow
Carbon
Carbon
Sickled
was
used
Dioxide
Sickled
(per
Reticulocytes
(per
via a.-gage
dioxide
160 RBC)
Reticulocytes
100 relics.)
mm.
L.J.
9.5
0
10.5
2.
10.0
9.C
10
W.B.
for
sickling.
to
found
0.0
0.0
4.0
2.0.5
2.1.0
69.5
‘3.5
64.3
2.0.0
90.0
‘7.5
87.5
0
15.0
2.
16.5
‘‘‘5
33.5
the
red cell
of the
76.0
‘4.5
93.5
removed
through
of
4.0
the saline cresyl blue blood
were
Reticulocyte
mersion
of
of
needle
fading
94.4
15.0
cc.
be as good
77.7
8.
a8.o
La-gage
slow
7.0
92..5
‘9.5
of blood
to
8i.o
2.0.5
samples
fixation.
due
One
45.0
2.
10
Small
and a
4.5
0
I0
J.W.
I
9.0
0.0
0.0
45.0
the
cell
and
sickle
the
reticulum
a fixative
suspension
and
The
had
standard
placed
sickle
blood
A a per
io per cent
on a slide
cells.
under
Only
was
9.5
67.8
89.6
immediately
cent
cells
into the inverted test tube.
oil with
an oiled
immediately
and
slip,
were
solution
had
and
counted
tuberculin
injected
in order
formalin
solution
a cover
aoo
2.7.3
introduced
under
to be done
in formalin.
as the
was
reticulocytes
counts
was
intervals
stopper.
0.0
4.5
13.0
mixture
at appropriate
rubber
0.0
less
into
to avoid
a count
because
was
saline
effect.
made
under
of the
for
inaccuracy
in normal
of a fading
syringe
formalin
time
was
A drop
oil
im-
factor
of
fading.
The
found
order
data
per
obtained
ioo
to calculate
are
RBC
was
the
shown
divided
percentage
in table
by
the
of sickled
i
.
The
number
number
of sickled
of reticulocytes
reticulocytes
in terms
reticulocytes
per
100
of total
RBC
reticulo-
in
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
JANET
cytes.
By comparing
these
figures
467
WATSON
with
those
for
the
percentage
of total
sickled
cells, it is evident
that the rate ofsickling
of the reticulocytes
rate of sickling
of the whole
red cell population.
A reticulocyte
is quite similar
in the sickled
is shown
in figure
i.
A possible
theoretic
are
an ordinary
smear
ing.
In
tube
with
chamber,
carbon
answer
examined
objection
is that
to
this
two
fact
abnormal
was
amount
into
and
the
sickle
cells
interferes
blood
an equal
introduced
minutes,
that
shape
objection,
after which
dioxide,
was
after
to the
the
not
the
completely
of saline
the test
sickled
with
to the
form
reticulated
in
supravital
sickled
stain-
in
the
test
cresyl blue, previously
aerated
tube under
oil. The blood
was
reticulocytes
were
found
to he
well
stained.
%:‘
#
_
FIG.
FIGURE
Meniscocvtes
1.
stained
with
\Vright’s
stain.
sickled
shape
to the
biconcave
developed
elongated
I
by carbon
dioxide,
are shown
in the
sickled
proce
Cells
disk.
Note
scsatthe
the
stained
with
process
sickled
crcsvl
of unsickling
reticulocytc
at the
ends.Theblackspotswith
Fio.
2.
bloc
and
with
transition
left.
immediately
Two
refractiletiontoi:rs
counter-
forms
are
the
from
crcsent
forms
have
artcfact
in photog-
raphy.
FIGURE
crescent,
These
sickled
In
all
quickly
Peripheral
2..
seven
elliptical
cells
three
blood
and
lack
smear
oval
with
sickle
used,
within
sickling
a few
blue
cresyl
cells
can
the bizarre shapes seen in Fig.
methods
reversible
stained
shaped
Wright’s
stain.
None
seen.
of the
Three
sickle
reticulocytes
cells
and
are reticulated.
i.
of all
seconds
and
be
upon
erythrocytes
and
admission
normoblasts
of oxygen
to the
was
system.
DISCuSSIoN
Direct
counting
of the
progressive
sickling
of reticulocytes
on
aeration
with
car-
bon dioxide
(table
i)
by means
of a gas test tube chamber
has shown
that
most
reticulocytes
sickle as well as the more mature
cells. However,
it was
noted
in this
and in the other
methods
used that
the reticulocytes
with
the largest
amount
of
reticulum
were
somewhat
mature
usually
slow.
cells
have
late
Although
a lower
in sickling.
this
oxygen
may
be
The
few
due
to
tension
normoblasts
the
threshold
possibility
for
observed
that
sickling,
the
there
were
also
most
im-
is an
a!-
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468
SICKLED
ternative
explanation
reticulum
may
The
ERYTHROCYTES
that
the
presence
mechanically
virtual
absence
IN
SICKLE
CELL
of a nucleus
interfere
with
of reticulated
or of a large
the
sickle
ANEMIA
sickling
cells
in the
ordinary
paradoxic
at first glance.
The sickle
cells seen in the fixed
ferent
from the ones produced
by in vitro
sickling.2’
6,
9
shaped,
elliptic,
or oval and do not have the long processes
tions.
When
viewed
directly
in a hanging
drop,
these forms
or more
nitrogen,
elongated
processes
at the pointed
and to revert
to their
original
going
on to the
should
normal
perhaps
that
have
lost
periods
of time
is
their
‘
from
vessels.
and Bibb2 who
in their pleural
Diggs
cells
blood
For
smears.
this
being
Good
of stained
(fig.
smear
The
seems
2.)
appear
former
are
dif-
crescent
seen in sickled
preparaare seen to send out one
with carbon
dioxide
or
with
oxygen
without
reason
these
It may
be that
abortive
sickle
they
are
cells
old
cells
kept in the sickled
shape
for long
evidence
for the role of stagnation
had three
or ascitic
It should
smear
blood
on aeration
on aeration
terminology.
‘
sickle
stained
forms.
a distinctive
their
‘elasticity’
in stagnant
blood
by
presented
irreversible
discoid
have
ends
shape
quantity
process.
patients
with
fluid, although
be possible
sicklemia
who
had
they had none in
to produce
these
crescent
forms
in vitro by keeping
cells in their sickled
shape for long periods
of time, but attempts
so far have been unsuccessful.*
It is a well known
fact that sickle cells are not seen
the
in
blood
since
the
occur
in
smears
oxygen
fixed
nation
with
of erythrocytes
sickled
perfused
young
cells,
shape
in vivo.
cell
increasing
reflected
have
Or
these
more
This
(i8
could
mm.
be
Hg)4
the
crisis
Ifwe
accumulation
in the peripheral
is not
surprising.
The
be an important
destruction
of the old
A personal
maintained
oxygen
.
patients
technic
or
preliminary
in the
those
showing
equilibration
and
so per
cent
carbon
Dr.
form
was
the
with
may
Tomlinson’s
predicted
would
never
and
and
the
the
trait
and
Shu
Chu
sterile
for
during
dioxide.
only
indicates
unable
incubation
hours.
also
that
cannot
be
of sickled
as well
that
This
to a gas
little
sickle
signifi-
their
could
abnorbe an
necessarily
among
being
various
of the
sickled
Thus,
a crisis results
of new young
red
as other
sterile
the
noted
cells”
and
that
there
fragility
of defibrinated
exposure
estimated
finding”
this
increase
diverse
reports
to reassume
twenty-four
continuous
the
are
entirely
fibrotic.
sickle cells differs
Emmel’4
mechanical
Shen
them
for stag-
pulp and
dragnet
in their final demolition.
cells and in the outpouring
renders
they
of time
Bibb2 and Smith’2
found
Shapiro3
found
that
the
onset.
to sickle
so that
surpass
had not become
of circulating
viscosity
of sickled
in organs,
so
appear
from
sickled
employed
after
increased
factor
sickled
reticulocytes
communication
rocytes
The
the
fell
forms
accept
of sickled
forms
without
blood.
If this is so, the
16 must
Although
and
‘
the length
of the spleen
be an important
that organ
the number
The high
sequestration
‘elasticity,’
‘
be that
it may
abnormal
patients
in whom
as to whether
before
investigators
cells2’
in the
trait.
sickling
to time in the same patient.
Diggs
while
Sydenstricker#{176}
and Murphy
increased
nitrogen
may
of the reticulocyte#{176}’
cant variation
in his patient.
mal shape
tend toward
their
mie
sickle
in vitro
cells are actually
stuck
in the interstices
out, it would
seem that the spleen could
from time
variation,
*
for
to prctduce
life span
erythrocytes
in those
There
is disagreement
cells.
the
necessary
being
in the sickled
five to six day
cells
persons
vivo.
Reticulocytes,
not
of
tension
cells,
incubation
discoid
blood
incubation
mixture
they
in vitro
form
samples
upon
of eryth-
exposure
from
was
carried
composed
of
are
either
out
90
to
aneafter
per cent
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
JANET
.
not
found
anoxic
in
the
organs,
irreversibly
sickled
irreversible
stagnation,
shape.
sickle
anoxemia,
forms
increasing
469
WATSON
As
the
appear,
sickling,
maturing
and
red
augment
thrombosis,
cell
the
and
stagnates
vicious
in
cycle
of
hemolysis.
SUMMARY
Data
I.
the
have
sickle
blood
slowly.
been
cell
cells.
presented
trait
The
or
most
cell
that
anemia
immature
Orthochromatic
2..
to show
sickle
most
were
the
shape,
cells
and
that
which
are
these
have
unable
forms
lost
their
to revert
are
to
so rarely
to
of
while
disk.
with
mature
red
to sickle
normoblasts
more
which
not be sickled.
stained
smears
stagnating
This
be reticulated
patients
more
tend
type
‘
a biconcave
found
normoblasts
only
‘elasticity’
‘
as do
types could
seen in ordinary
.
old
from
readily
and
sickled;
the basophilic
and polychromatophilic
3 It is suggested
that the sickle cell forms
sent
as
reticulocytes
normoblasts
reticulocytes
sickle
in
would
when
repre-
the
sickle
explain
stained
the
with
fact
brilliant
cresyl blue.
ACKNOWLEDGMENT
for
I wish
to express
many
stimulating
suggestions,
this
and
preparing
gratitude
my
report
to Dr.
William
to Dr.
to Mrs.
B. Castle,
John
Muriel
M.
Dr.
Pearce
MacDowell
William
Dock
(Department
(Department
of Medicine)
of Pathology)
(Department
of Pathology)
for
for the
assistance
in
photomicrog-
raphy.
REFERENCES
V. P.,
1 SYDENSTRIcKER,
in children
1*
with
J. B.,
SCRIVER,
AND
W. A.,
MULHERIN,
necropsy
in one case.
T. R.
WAUGH,
AND
Dis.
: Studies
R. W.: Sickle
HOUSEAL,
Am.J.
on
Child.
a case
26:
132.,
cell anemia:
Report
of two
cases
192.3.
of sickle-cell
anaemia.
Canad.
M.
J.
A.
23.’
375,
1930.
2
L. W., AND BIBB,J.
: The erythrocyte
in sickle cell anemia.J.
R. C., ANDSHAPIRO,
S.:The
pathology
ofsickle
celldiscase.
I. J. : The sickling
phenomenon,
with
special
reference
D,oos,
3 MURPHY,
4 SHERMAN,
trait.
Bull.
Johns
6 GRAHAM,
7 PONDER,
Biol.
Exper.
6-.’
309,
tomy.
AND
study
to the
L. E.,
Clin.
AND
W. J.
Path.
877,
25.’
J.
:
554,
24.’
A study
cell
anemia.
formation.
cell anemia.
LAWRENCE,
Investigation,
11 TOMLINSON,
376,
23
differentiation
5945.
of sickle
cell
1940.
: Sickle
ofsicklecell
V. P.: Sickle
9 SYDENSTRICKER,
E. B.
GILLESPIE,
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