Clinical Science (1983) 65,635-643
635
Importance of platelet-free preparations for evaluating
lymphocyte nucleotide levels in inherited or acquired
immunodeficiency syndromes
A. GODAY*, H. A. SIMMONDS*, D. R. WEBSTER*, R. J. LEVINSKYt,
A. R. WATSON$ A N D A. V. H O F F B R A N D I
*Purine Laboratory, Guy’s Hospital Medical School, London, tDepartment of Immunology, Institute for
Child Health, London, $Department of Paediatric Nephrology, Hospital for Sick Children, Ontario, Chnada,
and f Department of Haematology, Royal Free Hospital, London
(Received 18 Febmry/20 May 1983; accepted 14 June 1983)
Summary
1. Low ATP/ADP ratios have been reported
consistently for nucleotide levels of mononuclear
cells separated from peripheral blood by conventional techniques.
2. We have established that these low values
(mean 2.3 : 1) were not due to cell damage or poor
viability, but resulted from heavy platelet contamination, which is unavoidable when heparinized
blood is used. The results reflect the low ATP/
ADP ratios (mean 1.6: 1) characteristic of platelets. Platelet-free extracts from defibrinated blood
had very high ATP/ADP ratios (mean 17.4 :1).
3. The initial finding of detectable amounts of
deoxy-ATP and deoxy-GTP in mononuclear cells
from children with two distinct inherited immunodeficiency disorders [adenosine deaminase (ADA)
and purine nucleoside phosphorylase (PNP)
deficiency respectively] many have been due to
contamination by nucleated erythrocytes as well
as platelets in non-defibrinated preparations.
4. Defibrination before nucleotide extraction of
mononuclear cells from a patient with T-cell
leukaemic/lymphoma treated with the ADA
inhibitor deoxycoformycin enabled the demonstration of grossly raised deoxy-ATP levels relative
to deoxy-ADP levels (ratio 16.1 :l), associated
with severe ATP depletion. This reciprocal relationship between ATP and dATP was found by
us previously in the erythrocytes in inherited ADA
deficiency.
Correspondence: Dr H. A. Simmonds, Purine
Laboratory, Clinical Science Laboratories, Guy’s
Tower (17th and 18th Floors), Guy’s Hospital,
London Bridge, London SEl 9RT.
5. These findings underline the importance of
extracts uncontaminated by platelets, or nucleated
erythrocytes, in the evaluation of lymphocyte
nucleotide levels in inherited or acquired immunodeficiency syndromes.
Key words: adenosine deaminase deficiency,
deoxy-ATP,
deoxycoformycin,
deoxy-GTP,
lymphocyte nucleotides, platelet nucleotides,
purine nucleoside phosphorylase deficiency.
Abbreviations: ADA, adenosine deaminase; dCF,
deoxycoformycin; PNP, purine nucleoside phosphorylase.
Introduction
Few published studies include detailed information of nucleotide levels in leucocytes of the
peripheral blood [ 1-61. We recently investigated
nucleotide levels in both erythrocytes and socalled ‘lymphocytes’, separated by Ficoll-Triosil
from peripheral blood of immunodeficient children
with inherited disorders of purine metabolism
[7, 81. Consistently low ATP/ADP ratios were
found in the lymphocytes of both patient and
control cells, separated by the same technique at
the same time [7, 81. The same low ratios were
noted in a control series of nucleotide levels in
lymphocytes obtained from healthy men and
women by identical methods [9].
We have now extended these studies and compared nucleotide levels in platelets and lymphocytes from heparinized and defibrinated blood.
The results c o n f i i that the earlier low ATP/ADP
ratios were due to heavy platelet contamination,
unavoidable in lymphocytes separated by con-
636
A. Goday et al.
ventional techniques. Results obtained in immunodeficient children have been re-evaluated on this
basis and compared with data from a single patient
treated with the adenosine deaminase (ADA;
EC 3.5.4.4) inhibitor, deoxycoformycin.
Subjects
ControIs
Initially blood from 16 healthy control subjects
was collected into preservative-free heparin and
separated by routine methods with Ficoll-Triosil
(see [lo, 111). Subsequently, the blood from 13 of
the same control subjects was defibrinated before
separation. Peripheral blood mononuclear cells
separated by either technique were washed once
and the erythrocytes removed by hypotonic
lysis [3]. The importance of hypotonic lysis was
tested by omitting this step in duplicate analyses
in four instances. After two further washes the
mononuclear cells were re-suspended, counted and
diluted to a concentration of 1 x 106/ml. Viability
was tested by using trypan blue (exclusion >95%).
Contamination with granulocytes and erythrocytes
was minimal, but significant numbers of platelets
(not quantified) were present in the heparinized
blood preparation.
Patients
Mononuclear cells were separated from
heparinized blood from two immunodeficient
children (nos. 1 and 2) with ADA deficiency
[8, 91, both before and after erythrocyte exchange
transfusions, and one child (no. 3) with purine
nucleoside phosphorylase (PNP; EC 2.4.2.1) deficiency who never received a blood transfusion
[7]. Higher speeds were often required t o bring
down the mononuclear cells from these children
and the preparations almost invariably contained
a number of nucleated erythrocytes, which could
not be removed by hypotonic lysis [9].
Unfortunately, sufficient blood to obtain
platelet-free nucleotide levels was available only on
one occasion from the PNP-deficient child (the
child died subsequently of a parainfluenza virus
type 111 infection). By the time the importance of
platelet-free preparations had been established,
both ADA-deficient children had received bone
marrow transplants and were not investigated
further.
A single sample of defibrinated blood was
also obtained from a patient with T-cell leukaemic/
lymphoma on the last day of a 5 day treatment
period with deoxycoformycin (dCF), at a dose of
0.25 mg/kg given by bolus intravenous injection
daily for 5 days.
Methods
Preparation of platelet-free nucleotide extracts
Because of the heavy platelet contamination
inherent in the cells from heparinized blood,
different methods were investigated in attempts t o
produce a platelet-free extract. Two involved lowspeed centrifugation either before (i) or after (ii)
isolation of mononuclear cells by Ficoll-Triosil.
(i) Blood diluted in phosphate buffered saline
(PBS)/S% EDTA (2: 1 , v/v) was initially centrifuged at 130g for 15 min. The top layer, containing the plasma rich in platelets, was removed and
centrifuged for 10 min at lOOOg to sediment the
platelets. The supernatant was mixed with the
pellet of the first centrifugation. This step was
repeated once more and the mononuclear cells
were then separated by Ficoll-Triosil.
(ii) The second method consisted of dilution of
blood, as above, but followed immediately by the
Ficoll-Triosil step.
Mononuclear cells separated by either of the
above methods were then washed three times in
physiological saline by centrifuging at low speed
(5 min at 15Og) or subjected to three further
washes at higher speeds (5 min at 450g) before
counting and extraction. Platelets were still
evident microscopically in both preparations but
were not quantified.
(iii) A third method employing an initial defibrination step, using either glass beads or wooden
applicators (Macrom), was tried subsequently.
Heparinized blood was stirred vigorously at room
temperature with wooden applicators until an
adherent clot formed (2-3 min). This method
gave the most satisfactory results and normal
values for nucleotide levels were subsequently
established in defibrinated blood from 13 healthy
subjects as described in detail below.
Separation of peripheral blood mononuclear cells
(defibrinated blood)
Venous blood (20 ml) was defibrinated as
described above and diluted with a solution containing phosphate buffered saline (PBS)/5% EDTA,
pH 7.2, in the proporation of 2 : 1. Portions
(5 ml) were then poured into plastic tubes and
2 ml of Ficoll-Triosil, density (1.077 g/ml (Ficoll
400, Pharmacia, Uppsala, Sweden; Isopaque
440 mg of I/ml, Nyegaard A/S, Oslo, Norway),
was layered carefully underneath. The tubes were
Platelets and nucleotides in lymphocytes
then centrifuged at 450g at room temperature for
20 min. Mononuclear cells were collected from the
white opaque interface and washed three times
in physiological saline at 4°C (450 g for 5 min).
The washes included an erythrocyte lysis step
employing hypotonic shock, the cell pellet from
the first wash being initially resuspended in 1 ml
of isotonic sodium chloride solution (saline).
Water (3 ml) was then added and after no more
than 20 s isotonicity was immediately restored
by adding 1 ml of 3.5% NaCl.
After two further washings with saline, the
mononuclear cells were resuspended in a small
volume of saline, counted in a haemocytometer,
and the viability was tested by trypan blue
exclusion. Platelet contamination was minimal.
637
mated system was used to measure nucleotide
levels in platelets and platelet-free mononuclear
cell preparations. The h.p.1.c. system used for the
cells separated from heparinized blood has been
described in detail elsewhere [8, 91. For the
platelet-free extracts a Hichrom APSHypersil
column was used (5 /an, 25 cm x 4 m m internal
diameter) with a linear phosphate gradient
(gradient 6) at a flow rate of 2 ml/min, increasing
to 100% buffer B in 20 min. Buffer A contained
KH2P04 (5 mmol/l) at pH 2.65; buffer B contained KH2P04 (68g/l) plus KC1 (68g/l) at
pH 3.80. The same system was used for the platelet
extracts.
Results
Nucleotide levels in control subjects
Separation o f platelets
Table 1 compares nucleotide levels in mononuclear cells of healthy control subjects [ 9 ] , from
heparinized (n = 16) as distinct from defibrinated
blood (n =13). Platelet nucleotide levels in 10
healthy controls subjects are listed for comparison.
It is clear that the low ATP/ADP ratios obtained
from heparinized blood (mean 2.3) were due to
the heavy platelet contamination, confirmed by
microscopy. Platelet ATP/ADP ratios gave a mean
value of 1.6. By contrast, in the defibrinated
preparation (microscopically platelet-free) very
high ATP/ADP ratios were obtained (mean 17.4).
The ATP/GTP ratio was similar in all instances
(range 5.4-7.7).
The dramatic fall in mononuclear cell ADP
levels after defibrination was associated with a
Nucleotide extraction procedure
small but significant fall in ATP, possibly less than
Duplicate samples containing 1.5 -2.0 x lo6 would have been expected from the platelet
cells for mononuclear cells or 2-4 x lo* for plate- nucleotide levels. This difference could have been
lets, were centrifuged for 10 min at 450 g at 4OC. due to the rapid breakdown of platelet ATP to
The supernatant was removed by aspiration and ADP, which would have occurred during the
100 pl of ice-cold 10% trichloroacetic acid, con- Ficoll-Triosil separation at room temperature by
taining a radioisotope tracer (14C), was added to conventional techniques. Great care was taken
the pellet while being mixed gently on a Vortex. during the preparation of the platelet nucleotide
This suspension was centrifuged for 1 min at levels to avoid this by carrying out all procedures
12 OOOg (Beckman Microcentrifuge) and the super- at 4°C (see the Methods section).
natant immediately placed on ice. The trichloroacetic acid was extracted with water-saturated
ether to a pH above 5.0. The counts (14C) in 1 0 4 Establishment of conditions for reproducible
of extract were also calculated to correct for any mononuclear cell nucleotide levels
dilutional error (cell water, saline suspension,
Table 2(A) compares results with or without
medium etc.). Extracts were stored at -2OOC if hypotonic lysis of contaminating erythrocytes,
not analysed immediately .
and confirms the necessity of this step. Table
2(B) also demonstrates that despite extra washes
in both other methods employing low-speed
Measurement of nucleotide levels
centrifugation to remove platelets the ATP/ADP
A Waters Associates high pressure liquid ratios were still extremely low, consistent with the
chromatography (h.p.1.c.) trimodule fully auto- platelet contamination evident microscopically.
Samples of venous blood collected into 3.8%
sodium citrate solution were centrifuged very
slowly to allow sedimentation for 20min at
130 g at 4OC. The top layer containing the plateletrich plasma was diluted in physiological saline and
spun at 1600 g (3200 rev./min) in a refrigerated
MSE centrifuge for 30min. The platelet pellet
was washed twice with saline at 450 g (2000 rev./
min, MSE) for 10 min.
All steps were performed at 4 O C . Platelet
numbers were determined by counting in a Coulter
model ZF counter (Coulter Electronics, Harpenden, U.K.).
ABLE 1. Comparison
of nucleotide levels in mononuclear cells from heparinized or defibrinated blood, and platelets in healthy control s
lues for nucleotide levels in mononuclear cells, separated from (A) heparinized o r (B) defibrinated blood, compared with platelet lev
. The results demonstrate the low ATP/ADP platelet ratio and the effect of heavy platelet contamination on the ATP/ADP ratio of '
d from heparinized blood. (Ranges only are given for defibrinated blood and platelets because of the nonGaussian distribution
ated.) NAD', Nicotinamide-adenine dinucleotide; UTP, CTP, pyrimidine nucleotides; UDPS, UDP sugars. -, Below the limits of dete
method.
Nucleotide concentration (nmol/106cells)
ATP
ADP
AMP
GTP
GDP
AMP
NAD+
UTP
UPDS
CTP
3.07k0.70
1.35k0.37
0.18rO.06
0.40k0.10
0.23r0.07
-
*
*
*
*
2.88
1.44-4.45
0.17
0.09-0.27
0.03
0.01-0.07
0.46
0.11-0.87
0.06
0.04-0.12
-
0.16
0.03-0.37
0.37
0.17-0.45
0.13
0.06-0.29
0.08
0.04-0.18
lear cells
inized
(n = 16)
f SD
rinated
(n = 13)
-
Nucleotide concentration (pmol/106platelets)
ATP
ADP
AMP
GTP
GDP
AMP
NAD'
UTP
UPDS
CTP
95.72
55.66-137.19
59.10
35.3-17.2
7.70
2.7-12.3
17.71
7.9-33.2
9.90
3.6-17.1
1.81
0.75-4.6
4.10
2.26.7
7.92
3.7-14.1
2.98
1.1-4.5
1.99
1.1-3.8
n = 10)
antified because of poor resolution in the system used initially.
Platelets and nucleotides in lymphocytes
639
TABLE2. Nucleotide lewls in mononuclear cells separated from heparinized blood of control subjects:
importance o f erythrocyte lysis and platelet removal
Nucleotide levels (means of duplicate analyses) in four different subjects (nos. 1-4) showing: (A) the
effect of variable contamination with erythrocytes on nucleotide levels; (B) the low ATP/ADP ratios still
obtained despite low-speed centrifugation to remove platelets, before (B) (i), or after (B) (ii), FicollTriosil separation. a, No erythrocyte lysis; b, erythrocyte lysis step included. -, Below the limits of
detection.
Nucleotide concentration (mol/lO' cells)
ATP
ADP
AMP
GTP
GDP
GMP
ATPIADP ATPIGTP
0.42
0.39
0.34
0.37
0.35
0.30
0.31
0.25
0.19
0.18
0.21
0.18
0.12
0.17
0.14
0.11
-
2.4
2.5
2.4
2.2
2.5
2.1
2.8
2.3
7.6
7.6
7.3
6.6
5.9
7.0
6.6
5.6
-
3.9
4.4
-
2.8
4.3
-
-
6.4
5.3
3.3
4.2
4.4
4.7
-
2.0
1.3
1.9
4.6
5.2
4.9
(A) Nucleotide levels before and after erythrocyte lysis
3.18
2.97
2.49
2.46
2.05
2.11
2.04
1.40
1.3
1.19
1.02
1.11
0.83
0.98
0.72
0.60
0.13
1.14
0.16
0.10
0.14
0.12
0.17
0.11
(B) (i) Nucleotide levels after platelet removal before Ficoll-Triosil
X 3 washes at 15Og
1
1.84
0.47
0.33
1
2.02
0.72
0.16
0.42
0.17
X 3 washes at 450 g
0.46
0.02
(u) Platelet removal after Ficoll-Triosil
X 3 washes at 15Og
2
3
2.12
1.90
1.99
0.33
0.36
0.61
0.44
0.37
0.28
1
2
3
2.23
2.48
1.61
1.14
0.96
0;87
0.23
0.42
0.3 1
1
0.51
0.43
0.42
0.15
0.13
0.19
X 3 washes at 450g
For this reason the third method, using defibrinated blood, which gave the high ATP/ADP ratios
demonstrated in Table 1 (mean 17.4), was
adopted.
Patients
Mononuclear cell nucleotide levels in two
children with inherited ADA deficiency are shown
in Table 3. The cells were separated from
heparinized blood before (i) and after (ii) erythrocyte exchange transfusion [8]. The low ATP/ADP
ratios presumably reflect the high platelet contamination by this method. The high levels of
dATP and dADP in the preparation from patient
no. 2 before but not after transfusion, together
with the unusually high ATP levels pre-trans-
0.49
0.48
0.33
0.29
0.44
0.20
fusion, is attributed to contamination with
nucleated erythrocytes, which could not be
removed despite repeated attempts at hypotonic
lysis. It is noteworthy that this child had the
highest pre-transfusion erythrocyte dATP levels
yet recorded [ 111. The extract obtained posttransfusion contained ATP levels similar to the
control values and no detectable dATP, dADP or
nucleated erythrocytes, supporting the above
conclusion.
Extracts of mononuclear cells from heparinized
blood of the PNP deficient child contained low
but detectable amounts of dGTP on three
occasions [7], as demonstrated by the representative result in Table 3. By contrast dGTP was not
detectable in the last extract from defibrinated
blood, which was likewise the only extract which
did not contain macroscopic or microscopic
TABLE3. Nucleotide levels in mononuclear cells from immunodeficient patients
ntative results showing nucleotide levels in 'lymphocytes' from immunodeficient children separated by conventional methods: A, ei
after transfusion with packed irradiated normal erythrocytes for the adenosine deaminase (ADA-) deficient children [8, 91, as we
de phosphorylase (PNP-) deficient child who was never transfused [7]; B, cells were later obtained on one occasion from the PNP
ated blood. Results are compared with levels in defibrinated blood from a leukaemic patient treated with the ADA inhibitor deox
Note the similar values for dATP in patient no. 2 and the dCF treated patient, which contrast with the ATF' depletion in the latter, c
ed ATP levels in patient no. 2. (i) Before exchange transfusion; (ii) after exchange transfusion. A, Heparinized blood; B, defibr
-, Below the limits of detection for the method.
Nucleotide concentration (nmol/lO' cells)
ect
ATP
ADP
AMP
GTP
GDP
dATP
dADP
dAMP
dGTP
dGDP
ATP/ADP
ATP/GTP
3.34
3.36
0.98
1.33
0.16
0.10
0.46
0.57
0.07
0.21
-
-
-
-
-
-
-
-
3.4
2.6
7.3
5.9
0.93
0.76
0.91
0.49
0.09
0.29
0.31
0.1
0.92
0.82
0.35
0.32
0.22
0.25
-
1.22
0.17
-
-
(PNP') A
B
6.15
2.76
3.86
3.27
6.6
3.6
4.0
6.7
6.7
3.4
10.5
10.2
ith T-cellleukaemia
F
B
0.695
0.131
0.001
0.348
0.044
1.226
5.3
2.0
deficient children
(ADA-) A(i)
(fi)
(ADA-) A(i)
(fi)
-
-
0.076
-
-
-
-
-
0.21
-
-
0.1
-
-
Phtelets and nucleotides in lymphocytes
evidence of contamination with nucleated erythocytes. This suggests that the raised dGTP levels in
the heparinized blood preparations also derived
from contaminating nucleated erythrocytes
removed with the platelets during defibrination.
Although GTP levels in the PNP deficient cells
were within the normal range, GDP was undetectable and the ratio of ATP relative to GTP was
much higher than in controls or the ADA deficient
patients.
By contrast to the results in the ADA deficient
children, the mononuclear cell extracts from the
leukaemic patient treated with the ADA inhibitor
deoxycoformycin contained very high levels of
dATP and dADP, with an equally high dATP/
dADP ratio (16.1), but this was accompanied by
severe ATP depletion (Table 3). The mononuclear
cells were obtained 5 days after the instigation of
therapy, i.e. at the end of the treatment period.
Discussion
Problems associated with the accurate assessment
of mononuclear cell nucleotide levels in peripheral
blood have been noted previously by several
workers in the past decade. They considered that
the low ATP/ADP ratios found were due to cell
damage or the time taken in the method of separation used [l-41. The same low ratios were first
encountered by us during the investigation of
nucleotide levels in the so-called ‘lymphocytes’ of
immunodeficient children. Because of the severe
lymphopenia, very few cells (separated by conventional techniques) were available, since most
were required for assessment of immunological
factors. A preliminary report, which details these
and other difficulties, has already appeared [9].
The difficulties also included spurious peaks in
the chromatogram obtained by h.p.l.c., easily
mistaken for deoxynucleoside derivatives derived
from the Ficoll-Triosil used in separating the cells,
which could be removed by adequate washing. The
EDTA produced similar problems [9].
However, the most constant problem (despite
adequate washing, the inclusion of a very necessary
erythrocyte lysis step and attempts to speed up
the separation to improve cell viability), was the
very low ATP/ADP ratios obtained. The present
studies have confirmed that these low ratios are
not due to poor viability or the time taken for the
separation, but to heavy contamination with platelets. Furthermore, these platelets could only be
removed by defibrinating the blood first. With
this technique, much higher ATP/ADP ratios were
obtained (mean 17.4: 1, compared with 2.3: l),
results which are higher even than the 10: 1 ratio
64 1
characteristic of erythrocytes [7, 81. The very low
ATP/ADP ratios for platelets obtained from blood
from the same control subjects (mean 1.6: 1)
demonstrate the importance of platelet removal in
order to obtain meaningful results.
Identification of the pitfalls inherent in the
preparation of nucleotide extracts from heparinized
blood has enabled reassessment of nucleotide
levels in extracts of mononuclear cells from three
different immunodeficient children. The results
indicate that the raised dATP as well as ATP
levels found in the mononuclear cells of one of the
ADA deficient children [9, 111 were due to contamination with nucleated erythrocytes. The same
explanation could possibly apply to similar fmdings reported by others [lo, 12,131. The presence
of detectable amounts of dGTP in the mononuclear cells from our PNP deficient patient could
likewise be explained on this basis [7].
It is noteworthy that the GTP levels in the PNP
deficient child’s mononuclear cells appeared to be
within the normal range. However, the depleted
GDP levels, as well as the much higher ratio of
ATP to GTP (1O.O:l compared with control
6.3 : l), suggest that the severe guanine nucleotide
depletion noted in the child’s erythrocytes [7]
may be reflected in other cells of the peripheral
blood. The significance of these results remains
to be established through similar observations in
other cases.
We originally speculated [14] whether the
severe ATP depletion we noted associated with
dATP accumulation in the erythrocytes in inherited ADA deficiency [8, 11, 141 might also be
implicated in the lymphopenia and accompanying
immunodeficiency. The same reciprocal relationship between ATP and dATP was later noted in
erythrocytes of patients and animals treated with
the ADA inhibitor dCF, but this was accompanied
in addition by severe haemolytic anaemia [15, 161.
A reciprocal relationship between ATP and dATP
had already been reported in leukaemic blast
cells during dCF therapy [17]. Both subsequently
have been constant findings in dCF treated
patients. However, the magnitude of the ATP
depletion in the blast cells in this report (ATP
0.7 nmol/106 cells; dATP 1.2 nmol/106 cells)
suggests that both ATP depletion and dATP
accumulation may be implicated in the lymphopenia produced during dCF treatment.
Although the above situation involving blast
cells may not be comparable, our inability in most
instances to find significant amounts of deoxynucleotides in the peripheral blood mononuclear
cells from our immunodeficient children suggests
that these remaining cells must have been very
immature cells, incapable of accumulating deoxy-
642
A. Goday et al.
nucleotides. This is supported by the recent
finding of a complete absence of known T-cell
precursors, as well as mature T-cells in blood
from an 18 week ADA deficient foetus [18]. The
latter is in accord with earlier work indicating an
important role for ADA in the earliest stages of
T-cell differentiation [ 191. The combined results,
together with current studies by others, cast
doubts on current hypotheses implicating deoxynucleotide triphosphate inhibition of ribonucleotide Ieductase and DNA synthesis as the underlying
mechanism of lympho-specific-cytotoxicity [20].
They indicate involvement at an earlier phase of
the cell cycle [20] in inherited ADA deficiency
also [18].
The present studies have demonstrated that low
lymphocyte ATP/ADP ratios obtained by ourselves [9], as well as by many previous investigators [l-41, do not result from poor viability due to
the time taken or to cell damage during the
method of separation [4]. They are due to heavy
platelet contamination. It is also interesting to
speculate to what extent variable platelet contamination might affect other parameters of
‘lymphocyte’ function as measured in vitro. The
effect on ‘lymphocyte’ enzyme levels has recently
been noted [21]. The establishment of reliable
methods of separation, as well as the normal values
for a healthy control population, will allow
critical evaluation of nucleotide levels in mononuclear cells of peripheral blood in any future
studies in immunodeficient patients.
Acknowledgments
We are indebted to the Medical Research Council,
the Nuffield Foundation, the Leukaemia Research
Fund, and the Special Trustees of Guy’s Hospital
for supporting these studies.
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