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Concentration
of Fetal
Red Blood
Cells
From
a
Mixture
ofMaternal
and Fetal
Blood
by Anti-i
Serum-An
Aid to Prenatal
Diagnosis
of Hemoglobinopathies
Yuet
By
Fetal
red
from
Wai
blood
mixtures
differential
This
David
cells
of
with
be useful
Gabriel
concentrated
and fetal
agglutination
will
G. Nathan.
were
of maternal
method
nosis
Kan.
cells by
serum.
anti-i
for prenatal
diag-
when
blood
hemoglobinopathies
to detect
sickle trait
a reliable
method
developed,
it has
been
that
Marie
from
by maternal
cells. The method
is
except in very rare cases in which
the
maternal
nated
the
C. Crookston
obtained
red
fetus
cells
is
are
synthesis
in fetal
of sickle cell anemia1-2;
placental
heavily
strongly
con-
aggluti-
by anti-i.
in aborted
midterm
of sampling
fetal
suggested
and
taminated
practical,
EASUREMENT
OF HEMOGLOBIN
proposed
for the prenatal
diagnosis
been used
Although
Cividalli,
blood
has
this method
fetuses.2-3
blood
in utero
blood
may
has
not
be adequate
been
has
yet
been
for studies
of amino
acid incorporation
because
the rate of globin
synthesis
in fetal cells is
much
higher
than that in adult
cells.2
However,
extensive
contamination
by maternal
blood
remains
a potentially
significant
problem.
To overcome
this difficulty,
we have used a method
for separating
fetal cells from a mixture
of fetal and adult
cells
by agglutinating
them
with
the antibody,
MATERIALS
Fetal
blood
Blood
was
thalassemia.
From
was
obtained
obtained
from
the
Division
the Department
Service
and
the
the
california,
San
ronto.
Canada.
Hartford
Yuet
Calif
94/22
adult
of
Medical
Francisco
General
Professor
from
the
pital.
Jerusalem,
recipient
USPHS;
M.D.:
of Pediatrics,
Fellow,
Children’s
of an International
on temporary
Israel.
leave from
C.
Marie
and
and the Departjnent
Crookston,
and
Chief,
Division
Harvard
Hospital.
San
School,
Postdoctoral
Research
the Department
B.Sc.:
Assistant
Francisco,
Calif
University
General
Boston,
and
Hospital
Mass.
Harvard
Fellowship
Professor
of
To-
Cividalli,
Medical
Hadassah
San
Medical
Gabriel
(1 F05
of
Associate
of California.
Children’s
of Pediatrics,
94110,
of Toronto,
Hospital;
University
Center
Medical
16666 and by grants from
of Dimes,
the John
A.
of Hematology.
Medical
cell
the Hematology
Medicine,
University
Medicine,
sickle
Hospitat
02115,
Laboratory
July 29, 1973.
A M-05581,
AM
Foundation-March
Medical
Hospital
with
Children’s
Mass.
of Pathology,
Laboratory
at hysterotomy.
a patient
Boston,
Council
of Canada.
Service,
San Francisco
and
removed
from
Medicine.
School.
Pathology
Research
Hematology
fetuses
donors,
Department
Clinical
Pathology
G. Nathan,
Research
Mass.;
of Toronto.
and Clinical
David
Associate
Special
Boston,
15-20-wk-old
March
4, 1973; re vised July 16, l973, accepted
by USPHS
Grants
A M-05322.
A M-15913.
Foundation
of Boston,
Inc.,
the National
Calif
Center;
the
San
and
METHODS
of
normal
Harvard
Laboratories,
Francisco.
of Medicine
Francisco,
School,
TW-1
781-02)
University
Hos-
Pathology.
University
Canada.
1974 by Grune
Blood,
of
Medicine
cord
other
Hematology
of
AND
umbilical
mother,
Foundation,
and the Medical
Wai Kan, M. B., B.S.: Chief,
Professor
M.D.:
the
of Pediatrics.
Clinical
Departments
Submitted
Supported
the Medical
the
of
Center,
and
from
anti-i.4
& Stratton,
Vol. 43, No. 3 (March),
Inc.
1974
411
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412
KAN
Table
1.
Recovery
and
Concentration
of Artificial
Total CPM x 1O
Before Agglutination
Per Cent Fetal
14
Cells in
Initial Mixture
H
Maternal
(p)
(a)
Fetal
Blood in
Fetal
(c)
Percent
Per Cent Recovery
C
Maternal
Fetal
(in)
(d)
165.1
Agglutinated
Cells
(f)
(q)
76.9
100%
3.0
76.2
7.3
73.0
90.9%
20
100.9
64.4
2.7
44.1
2.7
68.5
86.4%
10
187.6
55.3
1.5
21.7
0.8
39.2
84.6%
387.3
51.3
1.7
13.1
0.5
25.6
75.0%
41.0
5
c
xlOO
a
“Agglutinated”
m=
cell
cells
sample
packed
red cells
patible
plasma
2 mg glucose.
fetal
and
cell
were then
suspension
Tables
I and
was pipetted
four
number
times
with
the buffer,
at 37’C,
of 3H leucine
(50
experiments,
washed
in saline.
the 3H and
Table
2.
cell
were expressed
Reproducibility
“C
leucine
to the
labels
were
reversed.)
were
was measured
vial containing
(0.9%
then
adult
cells;
before
in the
Cells
was
preparing
Adult
Cells
Agglutination
From
Method
mixture
before
for
Fetal Cells
In Agglutinated
Cells -
S
1
Adult
(AA)
2
Adult
(AA)
3
4
5
10
52.3
71.1
5
56.9
76.4
Adult
and Methods.
50.4
10
(S-ThaI)
See Materials
31.2
(AA)
(AA)
5
5
10
Maternal
Maternal
in
was cooled
counting
the
Mixtures
In Initial
Mixture
type)
a 20%
as follows:
a IO-pI aliquot
of each sample
0.5 ml water.
After the addition
of 0.5 ml
Percent
(Hemoglobin
to
was
shown
Source of
Expenment
and
added
incubation
proportions
was incubated
at 37’C
for 2 hr. The
England
Nuclear
Corp.)
was added
as counts per minute of 3H and “C.
Fetal
of
com-
leucine),
pCi/mmole)
NaCI)
made
milliliter
(without
(300
added
One-half
0.5 ml serologically
of 19 I-amino-acids
saline
and Methods).
buffer.
ml buffer,
was
mixtures
of Differential
Concentrating
0.5
of “C
with normal
30% hydrogen
peroxide,
the tightly
capped
vial
to room
temperature
and 10 ml Aquasol
(New
The results
of each
3 pCi
(see Materials
phosphate
containing
Ci/mmole)
four times
Fetal-adult
free cells
Krebs-Ringer
20 pmoles
preincubation
2. The uptake
of radioactive
label
into a liquid scintillation
counting
3H and “C activity.
of residual
in I ml of a mixture
against
for 2 hr. (In some
samples
a small
washed
30 pCi
-xlOO
pf+(100-p)m
included
10 mm
0
p1
q=
resuspended
dialysed
After
cells,
continued
d
-xlOO
b
was
was
0
0
1=
-
Each
104.3
80.0
0
(v/v)
Agglutination
Cells
Cells
14
H
Maternal
(b)
by Differential
AL.
1O
214.5
50
The
x
3
C
Fetal
100
the
Total CPM
Agglutinated
in
Cells
of Fetal
of Fetal and Maternal
Mixtures
ET
5
77.0
10
87.6
5
71.5
10
85.0
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CONCENTRATION
OF
For differential
volumes
lightly
to dislodge
and
sediment
of diluted
moval
ml distilled
fetal
5 mm,
and
and
chains
differential
relative
as previously
unlabeled
1 : 10; the cell
In order
solution
for 30 mm
After
were
the
described.2
mixtures
The
cells
were
for
3H and
preparation
were
agglutinated
whether
cells
from
and
mothers
temperature
the
cells,
recovery
cells,
by
radioactivity
to
3 ml
of adult
was
were
and after
cellulose
determined.
cells
and
calculated.
fetal cells, before
carboxymethyl
adult
re0.5
As a con-
in the
proportions
as before.
heterozygous
were agglutinated
by anti-i under the conditions
described
were tested with serum Den. The red cells of normal
adult
parallel.
centnifuallowed
agglutinated
The
and
3H leucine-labeled
analyzed
The
were
20
tapped
tapped
twice
more.
After
free cells) were lysed with
activity.
adult
separated
with
2 mm,
(2 1 ‘ C).
To the
and
residual
“C
mixed
agglutinates
of agglutinated
of chain
with
was
g for
at 300
by aspiration.
centrifuged
cells (and
ratios
mixed
preparation
tapping,
was
counted
cell
centrifuged
at room
last
removed
in the final
red
were
from
3H and “C leucine-labeled
procedure.
The
chains
were
fetal
to ascertain
was
proportions
trol
tubes
the cell-serum
mixture
as possible,
the agglutinated
were prepared
agglutination
experiment,
incubated
cells
of each
The
repeated.
the suspended
chromatography
I : 1 and
was
413
I volume
(Den).
and
tubes
CELLS
anti-i,
of anti-i
10 pl of this
their
Globin
the
of the
water;
cells,
with
dilution
anti-i
was added;
many
free cells
ofas
BLOOD
the agglutinates,
tapping
for
RED
agglutination
of a I-in-SO
gation
FETAL
for
above,
-thalassemia
or hemoglobin
the red cells of 62 selected
subjects
and
newborn
infants
S
patients
were
tested
in
RESULTS
The cells separated
procedure
with anti-i
q in Table
from the artificial
were predominantly
I, it can
(15-fold)
when
blood.
relative
When
the
enrichment
increased,
be seen
the
initial
that
mixtures
by the differential
fetal cells. By comparing
the degree
mixture
of enrichment
contained
the
of fetal
smallest
of fetal
cells
cells
proportion
initial
mixture
contained
a larger
proportion
was less but the actual
recovery
of fetal
as did the proportion
agglutination
columns
p and
in the separated
cells
was
greatest
(5%)
of fetal
of fetal cells,
cells (column
(column
the
f)
q).
Table
contained
2 shows
the results
of
only low concentrations
glutination
periment
When
with anti-i,
there
was a marked
enrichment
of fetal cells in each ex(including
one in which the adult donor
had hemoglobin
S-thalassemia).
the globin
chains
of fetal cells were analyzed
before
and after
aggluti-
nation
with
Table
3).
anti-i,
no change
in the
A disproportionate
elevation
maternal
of the
blood
agglutinated
found
in the
loss
ratio of beta
and unlabeled
cells
alpha-
were
and
further
experiments
in which
the cell mixtures
of fetal cells (5%-lO%).
After
differential
ag-
ratio
of beta
plus gamma
cord blood
similarly
beta-chain
to gamma
of alpha-chain
tests,
the cells
Table
of all 50 newborn
3.
Ratios
analyzed,
region,
and
normal
of Radioactivity
barely
detectable
no gamma-chain
normal
with
infants
Before
agglutination
agglutination
with
with
anti-i
anti-i
detected
shown
of Globin
Chains
radioactivity
radioactivity
subjects
reacted
hemoglobinopathies.
reacted
0
After
was
was
(see
by the
to alpha.
When
a mixture
of 3H-labeled
was subjected
to agglutination,
and the
detected.
(Data
not shown.)
As shown
in Table
4, the cells of two of 70 adult
with anti-i,
as did the cells of 14 of the 62 patients
these
chains
radioactivity
in Fetal
very
strongly.
Blood
0+-y
V
a
0.07
0.89
0.07
1.10
was
was
weakly
In
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414
KAN
Table
4.
The
Reactio
n With
Anti-i
of Re d Cells
From
Pat ients
Adult
Tested
Type
patients
With Anti-i
Weak
Strong
62
SA
16
3
0
SC
4
1
3
0
SS
2
1
1
0
CA
3
3
0
0
CC
1
1
0
0
6
0
$thal
27
3
Normal
adult
Normal
newborn
Agglutination
13
33
trait
ethaltrait
.
Negative
AL.
Hemoglobinopathies
Reaction
Number
Hemoglobin
With
ET
2
70
50
at 20#{176}
C by serum
Den. diluted
1
0
68
2
0
0
0
50
1 : 50.
DISCUSSION
These
results
show
that
differential
of concentrating
fetal red cells
example,
an initial
concentration
agglutination
in mixtures
of 5%
by anti-i
is an effective
containing
fetal and
fetal cells was enriched
differential
agglutination.
Such concentrations
of fetal cells
tection
of beta-chain
synthesis
in fetal cells against
a background
The moderate
loss of fetal cells during
the procedure
is well
enrichment.
Kleihauer-Betke
stains5
of
fetal
blood
show
that
the
means
maternal
cells.
six- to 16-fold
permit
accurate
of maternal
compensated
distribution
For
by
decells.
by the
of
hemo-
globin A in the developing
fetus is clonal.#{176}
The data in Table 3 in the present paper
indicate
that the treatment
with anti-i
did not cause
a selective
loss of fetal cells
containing
hemoglobin
A, since the beta to gamma
globin
chain radioactivity
ratios
remained
unaltered
by this treatment.
We conclude
that the i antigen
is
present
on fetal cells in which the switch from gamma-chain
to beta-chain
production has occurred.
The partial
loss of alpha-chain
activity
is not explained
at
present,
but this loss is not important
when the object is to detect beta-chain
abnormalities.
Increased
maternal
synthesis
of hemoglobin
F during the first trimester
of pregnancy has been described.7
In the present
study, the gamma
chains of hemoglobin
derived
by chromatography
from the agglutinated
cells in a mixture
of 3H-labeled
maternal
cells and nonlabeled
fetal cells were not detectably
radioactive.
Thus,
since
maternal
cells containing
fetal hemoglobin
were not agglutinated
by this
particular
example
of anti-i, they did not interfere
with the detection
of hemoglobin
A synthesis
in the fetal
cells.
hematologic
disorders,
particularly
are strongly
agglutinated
by anti-i.8 The results
small increase
in i antigen
is occasionally
found
or $-Thal
gene, but that the agglutinability
of
less than that of fetal cells.
Thus the differential
agglutination,
method
of
plied to samples
obtained
by placental
aspirate,
In some
(1)
Estimate
(2)
Incubate
the percentage
of fetal cells
cells with radioactive
amino
thalassemia
major,
adult red cells
shown in Table 4 suggest
that a
in patients
heterozygous
for the S
these cells by anti-i is consistently
concentrating
in the following
by the Kleihauer-Betke
acid.
fetal cells can be apmanner:
technique.5
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CONCENTRATION
(3)
OF
FETAL
samples
When
RED
are
BLOOD
CELLS
found
415
to have
significant
maternal
cell
contamination,
separate
fetal cells by differential
agglutination
with anti-i.
(4) Test a sample
of the mother’s
blood with anti-i, in parallel
with the placental
sample.
If the maternal
i antigen
is greatly
increased,
it may be difficult
to separate
fetal cells using
anti-i.
In some
such cases,
a difference
in the ABO
groups
of
mother
and
fetus
glutination
can
of fetal
be exploited
by using
anti-A
or anti-B
for the differential
ag-
cells.
ACKNOWLEDGMENT
We are indebted
Tilley
to Dr. D.H.
for technical
Cowan
for anti-i
serum,
and to Miss Andr#{233}e
Dozy and Mrs.
Christine
assistance.
REFERENCES
I.
Hollenberg
MD,
Kaback
MM,
5.
Kazazian
HH:
Kleihauer
Adult hemoglobin
synthesis
by reticulocytes
from human
fetus
at mid-trimester.
Science
174:698, 1971
2. Kan YW, Dozy AM, Alter BP, Frigoletto
onstration
FD,
the
in
Nathan
the
DG:
human
diagnosis
287:1,
3.
Sickle
fetus:
of sickle
Potential
cell
for
anemia.
cell
gene
intrauterine
N
EngI
J
HH,
cell
7:200,
Kaback
MM,
hemoglobin
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Marsh
eines
35:637,
6.
Pediatr
WL:
1961
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in
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an
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a cold
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K: DemErythWochenschr
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in den
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1967
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1972
the Ii relationship
matol
of the sickle
rocyten
7.
Kazazian
trimester
4.
Detection
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von
WS:
mid-
antibody
defining
cells.
Br J Hae-
ME,
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of
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2 1:355,
8.
1972
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of red
Giblett
cells
major
and
Nature
(Lond)
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hemoglobin
ER,
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F in
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Br
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by anti-i
other
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Agglutinability
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J
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1974 43: 411-415
Concentration of Fetal Red Blood Cells From a Mixture of Maternal and
Fetal Blood by Anti-i Serum−−An Aid to Prenatal Diagnosis of
Hemoglobinopathies
Yuet Wai Kan, David G. Nathan, Gabriel Cividalli and Marie C. Crookston
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