Download Simplifying Lymphocyte Culture and the

CLIN. CHEM. 2611, 72-77(1980)
Simplifying Lymphocyte Culture and the Deoxyuridine Suppression Test by
Using Whole Blood (0.1 mL) instead of Separated Lymphocytes
Kshitlsh C. Das, Cathy Manusselis, and Victor Herbert
describethe use ofsmallvolumes (0.1mL) ofwhole
of the de novo pathway of thymmne-DNA synthesis. In the
blood instead of separated lymphocytes, in a microscale
conventional
procedures,
lymphocytes
are separated
from
technique of lymphocyte culture. We incubated at 37 #{176}C whole blood by use of various macromolecular gradients or by
filtration through a cotton or nylon column. These time0.1 mL of heparinized blood in 0.9 mL of 60 mmol/L
consuming processes entail loss of lymphocytes
and require
tris(hydroxymethyl)methylamine-buffered
Hanks-Eagle
about 50 mL of blood, characteristics
that limit the clinical
medium (pH 7.4)unsupplementedwithfetal
calfserum.
application of the procedures and preclude their use in pediIncorporation of [3H]thymidine or [125l]deoxyuridine
into
atric patients. Here we describe a microscale technique for
the DNA of 2 X i0 to 3.5 X iO lymphocytes was greatest
lymphocyte
culture in which whole blood (0.1 inL per culture
with 10-20 ug of phytohemagglutinin-P
per culture tube.
tube) is used.
We
Radionucleoside
phocytes on day
incorporation
peaked in normal lyma day later than in culture of sep-
4 (i.e.,
arated lymphocytes) and on day 8 in lymphocytes from
cases of chronic lymphocytic
leukemia. Lymphocyte
transformation in whole-blood culture was qualitatively
similar to that in cultures of separated lymphocytes from
the same person. Five to 10 mol of nonradioactive deoxyuridine
per culture was needed to suppress to about
10% of control the incorporation of subsequently added
[3H]thymidine or [125l]deoxyuridine into DNA. Shortening
the incubation to 1 h did not significantly affect qualitative
results
of the deoxyuridine suppression test. Whole-blood
culture
issimple, requires less blood, eliminates the need
to separate the lymphocytes,
studies based on lymphocyte
to routine clinical laboratories
technique is suitable for use
deoxyuridine suppression test
folate status.
and thus makes diagnostic
transformation
accessible
and pediatric patients. Our
in a simplified lymphocyte
to evaluate vitamin B12 and
AddItIonal Keyphrases:
microchemical
techniques
evaluation of vitamin 812 and folate status
pediatric chemistry
phytohemagglutinin-P
mitogen-stimulated transformation of lymphocytes
-
-
.
Mitogen-stimulated
transformation
of blood lymphocytes
in culture has been extensively
used for cytogenetic,
immunological, and metabolic studies. Data are commonly quantitated by measuring incorporation of a radioactive nucleoside
(e.g., [3H]- or [14C]thymidine)
into newly
synthesized
DNA.
PHA-stimulated’
culture of lymphocytes has also been used
as a model system for measuring
cellular folate uptake, and
for the dU suppression test(1-3), which measures the efficacy
Hematology
and Nutrition
Laboratory,
Veterans Administration
of Medicine,
Medical Center, Brooklyn,
Medical Center, Bronx, NY 10468, and the Department
State University of New York, Downstate
NY 11203.
A brief report of this work was presented at a meeting of the
American Federation for Clinical Research (Das, K.C., Manusselis,
C., and Herbert, V., A simplified
lymphocyte
dU (deoxyuridine)
suppression
test which can use finger-stick peripheral blood. Clin.
Res. 26, 618A (1978)].
Received May 10, 1979; accepted Sept. 26, 1979.
72
CLINICAL CHEMISTRY. Vol. 26, No. 1, 1980
Materials and Methods
Whole-Blood Culture
Blood was collected from apparently
healthy volunteers into
sterile, silicone-coated
heparinized
“Vacutainer
Tubes”
(Becton-Dickinson and Co., Rutherford, NJ 07070; Vacutainer
Tubes no. 3208KA, containing
357 USP units of sodium
heparin per 20 mL tube of blood; proportionately
less heparmn
is used for smaller amounts of blood). A 0.5-mL aliquot was
removed for total and differential
leukocyte counts, from
which the total lymphocyte count was determined.
The remaining heparmnized blood was diluted 10-fold with Trisbuffered Hanks-Eagle
solution (60 mmol/L, pH 7.4) containing 200 units each of penicillin and streptomycin
per
milliliter. One-milliliter aliquots containing 0.1 mL of whole
blood and 0.9 mL of Tris-Hanks--Eagle
medium (4) (Microbiological Associates, Walkersville,
MD 21794) were dispensed
into 10-mL sterile, silicone-coated
Vacutainer Tubes. The
contents of a 50-mg vial of Bactophytohemagglutinin-P
(PHA-P; Difco, Detroit, MI 48232), was dissolved in 5 mL of
THBSS (Microbiological Associates).
This 5 mL was further
diluted with THBSS to make up aliquots of different dilutions
of PHA-P; these were stored at -20 #{176}C.
At the time of use, the
aliquots were thawed, and 0.1 mL of each dilution was added
to the culture. Unused PHA solutions, once thawed, were
discarded.
Cultures were set up in triplicate and incubated at 37#{176}C
for
periods ranging up to five to six days. We evaluated lymphocyte transformation
by measuring incorporation
of [3H]TdR
or [‘251]UdR into DNA. A 3-h pulse with 1 Ci (50 iL) of
[3H]TdR (sp acty, 23 Ci/mmol; Amersham Searle, Arlington
Heights, IL 60005) and 1 1zCi (50 L) of (‘25I]UdR (sp acty, 2
Ci/L; Amersham Searle) was given to two separate sets of
triplicate cultures from each sample of blood.
I‘251]Deoxyuridmne may deteriorate unexpectedly. Before
each use, add 50 iL of [1251]UdR to 0.5 mL of a 250 mgIL solution of hemoglobin-coated
charcoal and 1 mL of TrisRanks, shake the mixture briefly, and centrifuge (5).If less
1 Nonstandard abbreviations
used: PHA, phytohemagglutinin;
13H]TdR, (3Hjthymidine;
[‘H]UdR, [3H]deoxyuridine;
Tris,
tris(hydroxymethyl)methylamine;
Salt Solution; and dU, deoxyuridine.
THBSS,
Tris-Hanks
Balanced
than 90% of the radioactivity
is adsorbed
onto the coated
charcoal, the material should be considered
deteriorated,
and
discarded.
After incubation, the reaction was stopped by adding 5 mL
of cold isotonic saline. Then cells were centrifuged
at 4#{176}C
at
2000 rpm for 10 mm, and the supernatant
fluid was discarded.
The erythrocytes
were shock-lysed
by adding 4.5 mL of cold
distilled water, followed by 1.5 mL of a 35 g/L NaCl solution.
This was centrifuged (2000 rpm, 4 #{176}C,
15 mm) and the supernate was discarded.
Five milliliters of a 30 mL/L acetic acid
solution
was then added to the pellet, vortex-mixed,
and
centrifuged (2000 rpm, 15 mm). The pellet was then washed
once with 5 mL of cold isotonic saline, the supernate discarded, 5 mL of cold trichloroacetic
acid (100 g/L) added to
the pellet, the mixture again centrifuged, and the supernate
discarded. “Soluene 100” (Packard Instruments,
Downers
Grove, IL 60615), 0.4 mL, was then added to each pellet and
the dissolved pellet was then washed into a scintillation vial
with 10 mL of “Instagel” scintillation
liquid (Packard). The
radioactivity
was measured with a Packard liquid-scintillation
counter, and disintegrations per minute (dpm) were measured
with quench correction
by use of an external
standard.
Cultures
in which [‘25IJUdR was used as the tracer nudeoside were processed
in a slightly
different,
but simpler
manner.
After shock-lysing
the erythrocytes
with distilled
water, followed by the addition of saline (35 g/L) solution, we
washed the pellet once again with 5 mL of cold isotonic saline
and added five drops of salt-free albumin solution and 5 mL
of the trichloroacetic
solution
(100 gIL). This mixture was
centrifuged
(2000 rpm, 4 #{176}C,
10 mm), the supernate
decanted,
and the radioactivity
of the pellet measured in a Packard
“Autowell” gamma counter. During the course of standardization of the microculture
technique,
we used volumes of
whole blood ranging from 50 to 300 jzL, diluted with TrisHanks-Eagle
medium to a final volume of 1 mL per culture
tube. One hundred
microliters
Tris-Hanks-Eagle
medium
of blood diluted
was the optimal
10-fold with
quantity,
i.e.,
showed maximum radionucleoside
incorporation.
Furthermore, use of a larger volume of blood in the culture makes
processing for DNA extraction
technically
difficult and
quenching
of radioactivity
greater; repeated
treatment
with
acetic acid (30 mL/L) to remove hemoglobin
presumably
also
results in loss of damaged nuclei and thus of DNA, decreasing
apparent
nucleoside
incorporation.
Table 1. Results of Deoxyuridine (dU)
Suppression Test In Whole-Blood Lymphocyte
Cultures (with PHA-P) from 10 Normal Subjects
E3H]TdR
[125I]UdR
Incorporation
Into DNA after
Incubation for
3h
lh
Incorporation
Into DNA after
Incubation for
3h
7.2±
dU
2.5
9.3 ±
1.9
7.5 ±
2.3
8.2 ±
2.5
dU + MHF
dU + B12
dU + MHF +
B12
MI-F, 5-methyItetrahyofoIate:
after
addition
% of controls
8.0±
8.6±
1.9
1.8
7.8 ±
8.4 ±
1.8
2.5
9.0 ±
9.1 ±
2.4
1.6
8.8 ±
8.3 ±
2.1
2.4
B,2, hy&oxocobalamln
9.2 ±
1.4
9.0 ±
1.7
8.6 ±
2.5
9.3 ±
1.8
form: lnctiation. time
of radionucleosides.
dine (102 to i0
mol
DNA of subsequently
whole-blood
per culture tube) on incorporation
into
added
[3H]TdR
or [‘25IIUdR
in
cultures
and in separated
lymphocyte
cul-
tures.
Cultures
were pre-incubated
with various concentrations
of dU, with and without 5-methyltetrahydrofolate
and (or)
vitamin B12 (in hydroxocobalamin
form) for 1 h, followed by
a 3-h incubation
with 1 tCi of [3HJTdR or [125I]UdR on day
4 and 5, respectively
(corresponding
to the days of peak PHA
response).
The reaction was then stopped by adding 5 mL of
cold isotonic saline, and the subsequent
washing and extraction of DNA were as described above. As previously described
(1,3,6, 7), dU suppression
is expressed
as the percentage
of
incorporation
of [3H]TdR
or [1251]UdR into DNA
after
preincubation
with dU, compared
with the incorporation
of
these nucleosides
in replicate cultures (controls)
to which no
dU was added.
We found (Table 1) that dU suppression
of incorporation
of subsequently
added [3H]TdR or [1251]UdR into DNA during 1 h of incubation
with radionucleoside
was similar to that
obtained in the earlier procedure,
incubation was 3 h (3, 6, 7).
in which the duration
of
Lymphocyte Culture
Lymphocyte
culture
was set up by a modification
of the
method previously
described
(3). We cultured 2.0 X j#{216}5
to 3.5
x i05 lymphocytes
in 1 mL (final volume) of Tris-HanksEagle solution containing
100 to 150 mL of autologous serum
or fetal calf serum per liter of final medium. We used [3H]TdR
and [125IJUdR as tracer nucleosides
with a short (3-h) pulse
period, then terminated
the cultures
and divided them into
two separate sets of triplicate
cultures. These were processed
as described
above for whole-blood
cultures,
with respective
use of the two nucleoside
tracers.
Blood Samples
Blood was drawn from 10 clinically
healthy subjects (voland ourselves) who had normal values for serum vitamin B12, serum and erythrocyte
folate, serum iron, and
unteers
iron-binding
capacity;
persegmentation.
The
ranged from 43 to 48%,
leukocyte
counts from
cyte counts from 2000
the blood showed no granulocyte
hyhematocrits
of the blood samples
hemoglobin
from 140 to 155 g/L, total
7000 to 10500/mm3, and the lymphoto 3500/mm3.
dU Suppression Test
We compared
the effect
of pre-incubation
with deoxyuri-
Results
Whole-Blood
PHA -response.
Culture
Figure
1 illustrates
the incorporation
of
(3H]TdR
and [125IJUdR into the DNA of lymphocytes
in
whole-blood culture when different concentrations
of PHA-P
were used in 10 individual experiments with 2.0 X 10 to 3.5
X 10 lymphocytes
per culture tube. Incorporation of labeled
nucleosides was greatest with 20 tg of PHA-P per culture
tube; the responses to 10 and 40 tg per culture were also high
and were very similar on day 4. With 40 g of PHA-P there
was significantly
greater
clumping
of erythrocytes,
which
caused difficulty in washing the cells and in removing hemo-
globin from them during extraction
of DNA. Removal of he-
moglobin
is essential when [3H]TdR is used as the tracer nucleoside, but not when [1m1]UdR is used. However, inadequate
washing of the cells increases nonspecific radioactivity
because
free tracer nucleosides
are trapped
in the cell clumps.
The optimum
dose of PHA-P that gives peak transformation may vary slightly (10-40 tg per culture) with different
batches of the lectins, and it is advisable
to determine
the
dose-response
with every batch.
Time-response
curves. We ran dual triplicate samples with
CLINICAL CHEMISTRY, Vol. 26, No. 1, 1980
73
PHA-P
(0) UNDILUTED(1mg/culture)
(I) 1:10 DILL?T10N(iOO4ug/cullure)
(2) 1:25 DiLUTION(404ug/cuitur.)
(3) :50 DILUTION(2Opg/culture)
(4) 1:100 DlLUTl0N(l0ug/cuItur.)
(5) 1:200 D1LUTION(Spg/cuiture)
0
I-
(6)1:400
DILUTi0N(2.5ug/cuiture)
PHA-P
Wr)
ZI
x
80
>-E
Xo.
>-o.
‘V
I-
(0) UNDILUTED (Img/&IItur.)
11)1:10 OLUT1ON(IOOpg/cultur.)
(2) 1:25 DILUTION(4Opq/cuitur.)
(3) 1:50 DILUT10N(20ug/cuItur.)
(4)1:100 DILUTION(IOMQ/cultur.)
(5) 1:200 DILUTION(Spg/culture)
(6) 1:400 D1LUT1ON(2.Spg/cuiture)
(3)
(4)
(3)
60
(2)
ow
z
(4)
(2)
04O
(3)
(6)
0
I))
20
20
(0)
I
2
3
4
5
DAYS OF CULTURE [WHOLE BLOOD]
I
I
I
I
2
3
4
5
DAYS OF CULTURE
[wHoLE
BLOOD]
FIg. 1. Incorporation of [3H]TdR (left) and [1251]UdR (right) into DNA in whole-blood culture with various concentrations of PHA-P
(2.5-1000 sg per culture) on different days of culture
each of 10 blood specimens obtained from 10 different donors,
and studied with incorporation
of 13H]TdR and [I]UdR
into
DNA on different days of culture. With all 10 specimens, incorporation of the radionucleosides
was greatest
on day 4
possible
presence
of thymidine
phosphorylase
(EC 2.4.2.4).
In the above experiments,
we used sera from the same source
throughout
(Reheis Chemical Co., Armour Pharmaceutical
Co., Kankakee, IL 60901; batch 471506). We avoided the
(Figure 1) of culture.
As expected, incorporations
of
into DNA in PHA-stimulated
cultures were comparable
(Figure 1). The mitogen
response
of
the whole-blood culture was not significantly
affected for
lymphocyte
counts of 2 X 106 to 3.5 X 106 per milliliter of
Use of labeled
nucleosides.
PHA-P
(1)1:10
[3HJTdR and [‘I]UdR
blood.
The harvesting
procedure
with the acetic
described gave consistently
satisfactory
results
in the triplicate
Variables
of radionucleosides
(6)1:400 DILUTION(2.Spg/cullurs)
140
120
into DNA was usually
of autologous
serum, in eight of 10 experiments.
Fetal calf serum treated with hemoglobin-coated
(a procedure
similar to dialysis)
had a variable
[3H]TdR incorporation:
and
DILUTION(IOpq/cullure)
(5) 1:200 DIWTl0N(5/cultuve)
(4)1:100
160
in Both Culture Media
sometimes
it was sometimes
no
of
increased.
charcoal
effect
26, No.
I-
((5)
(2)
80
ow
60
CI)
/
40
20
/
‘I
S
(6)
/
on
normal, sometimes
We observed
similai
variations when cultures of lymphocytes or whole blood were
set up with autologous sera that had been treated with hemoglobin-coated
charcoal. The cause(s) of the variability is
unknown, but possibilities include (a) variation in lymphocytes among donors; (b) variable presence of thymidine and
other nucleosides and deoxynucleosides
in serum; and (c)the
CLINICAL CHEMISTRY, Vol.
I00
en-
serum added to appropriate
volumes of Tris-Hanks-Eagle
medium to give a final volume of 100 mL) inhibited 13H]TdR
incorporation
into DNA by 10 to 60%, compared with addition
74
(3) (:50 OILUTION(2Opg/cullurs)
cultures).
hanced by 6 to 15% over that in replicate
cultures
with
supplementations.
Fetal calf serum (i.e., 10 to 15 volumes
decreased,
(2) (:25DLUTION(4Opg/culture)
acid step we
(CV of 10%
Effect of supplementing
the culture medium
with autologous or fetal calf serum.
When the culture medium
was
supplemented
with autologous
serum (10 parts of autologous
serum added to 90 parts of Tris-Hanks-Eagle
solution),
in-
corporation
LUTI0N(IO0pq/cullure)
1, 1980
2
3
DAYS OF CULTURE
CSEPARATED LYMPHOCYTES]
Fig. 2. [3H]TdR incorporation
into DNA in separated lymphocyte
cultwes wftti various concentrations ofPHA-P (2.5-1000 rg per
culture) on different days of cultures
CULTURE OF SEPARATED LYMPHOCYTES
BLOOD CULTURE
WHOLE
0
.I00
0
O,4
04.
0
1-4
60
:EZ
oz
0
z
40
Ow
4c:l
cr
a‘-‘0
a->-
20
x
00
ow
Z0
I0
(,J
I’)
I0
10-2
100
[1251]UdR
I0-
102
NON-RADIOACTIVE
NON-RADIOACTIVE DEOXYURIDINEADDED
(pmoles/culture)
Fig. 3. Suppression of [3H]TdR or
i06
101
100
10’
DEOXYURIDINE ADDED
(pmoles/culture)
incorporation into DNA by added deoxyurldine (dU) in whole-blood culture (left),
and
cultures
ofseparated
lymphocytes
(right)
Mean and standard devIation of five different subjects (difference of [3H]TdRand [125I]1J
problem
by using neither fetal calf serum nor charcoal-adsorbed autologous
serum.
PHA -dose-response
and peak-time
response.
These two
variables
were compared
in five individual
experiments
with
cultures of purified lymphocytes
and whole-blood microcultures from the same donors. The concentrations
of lymphocytes in both types of cultures were similar (ranging from
2.0 X 10 to 3.5 X 10 lymphocytes per culture). Incorporation
of [3H]TdR or [‘25I]UdR was greatest with 20 rg of PHA in
purified lymphocyte cultures, as in the microcultures of whole
blood, but the peak-time response occurred on day 3 (72 h) of
40
120
COO
I-
(I)
80
2 60
ao 40
(I)
CLL (2)
20
0;
23456
8
9
DAYS OF CULTURE
Figure 4. Delayed DNA synthesis peak in mitogen-stimulated
chronic lymphocytic leukemia lymphocytes in whole-blood
culture
CLL-1 Is a 55-year-old man with hemoglobin = 105 gIL,packed cell volume =
33%, total peripheral blood leticocytes =75 000/nm3 (91% lymphocytes, 8%
neutrophils, 1% eosinophlls); CLL-2 isa 88-year-old man with hemoglobin =
115 g/L, packed cell volume = 34%, total peripheral blood leukocytes
58 500/mm3 (88% lymphocytes, 12% neutrophlls)
incorporationnot significant.
t < 0.05)
culture, a day earlier than in the microculture
(Figure 2).
of whole blood
Effect of pre-incubation with leoxyuridine on [3H] TdR
and [1251] UdR incorporation
into DNA (dU suppression).
As we expected on the basis of previous studies (1, 3, 7), pre-
incubation
with different concentrations
of deoxyuridine
decreased incorporation
of subsequently
added [3HJTdR or
[‘I]UdR
into DNA in the whole-blood culture (Figure 3, left)
(20 jzg of PHA-P per culture tube). Addition of 5-10 imol of
deoxyuridine
per culture tube suppressed
[3H]TdR or
[‘251]UdR incorporation
into DNA to about 10% of that for
controls, i.e., replicate cultures to which no deoxynridine was
added. The results of these experiments
with whole-blood
culture were similar to those with purified lymphocyte cultures from the same donors (Figure 3, right). The lymphocyte
count in both types of culture ranged from 2 X 105 to 3.5 X 10
cells per culture tube. The dU suppression of [3H]TdR or
[‘251]udR incorporation into DNA was similar after 1-h and
3-h incubation with radionucleoside.
Addition of 5-methyltetrahydrofolate
or vitamin B12 to the cultures had no further
effect on the degree of dU suppression in the 10 normal blood
samples (Table 1), as reported in previous studies (1, 3, 19).
The application of our procedure to the study of vitamin B12
and folate deficiencies will be described in a subsequent
communication.
Chronic Lymphocytic Leukemia
As shown in Figure 4, in whole-blood cultures, incorporation
of isotope into DNA is greatest on day 8 in lymphocytes
from
cases of chronic lymphocyctic leukemia, compared with day
4 in nonmalignant
lymphocytes.
Discussion
Lymphocyte
ological
states
transformation
has been studied
in vitro in normal and pathby numerous
using various culture techniques (8-20).
gested that the presence of erythrocytes
investigators
Earlier studies sugand granulocytes in
CLINICALCHEMISTRY.Vol. 26. No. 1, 1980 75
PHA-stimulated
lymphocyte culture may not significantly
impair the incorporation
of [3H]TdR into DNA (9, 12). On the
contrary, responses to antigens may in fact be higher in lymphocyte cultures containing other leukocytes than in cultures
of separated and purified lymphocytes (13). A major problem
concerning immunological and metabolic studies in cultures
of purified lymphocytes is that much blood is needed. Our
procedure requires only 0.1 mL of whole blood per culture
tube. The appropriate
culture conditions, dose-response
to
‘PHA, and peak-time
response
of radionucleoside
incorpora-
tion have been determined, and our results compare well in
consistency, reproducibility,
and magnitude with those obtained with cultures of purified lymphocytes from the same
donors. The method is simple, requires relatively little blood,
avoids time-consuming
lymphocyte separation and purification, and involves minimum manipulation.
This makes it
possible to use heel- or finger-prick blood from pediatric patients.
A crucial first step in the activation of lymphocytes by a
mitogen is the binding of the mitogen to the lymphocyte
cell
surface, and a quantitative
relationship exists between mitogen binding and subsequent blast transformation
(14). This
was evident in the present studies, because incorporation
of
[3HITdR or [‘25IJUdR was greatest with 20 g of PHA-P;
addition of more or less of the mitogens resulted in suboptimal
activation, both in whole-blood and purified lymphocyte
culture. However, the peak-time response in whole-blood
culture occurred a day later (day 4) than in cultures of purified
lymphocytes.
In the present study, supplementation
of the culture medium with 100 mL/L fetal calf serum usually decreased radionucleoside incorporation into DNA. On the other hand, fetal
calf serum reportedly
causes spontaneous
stimulation
of
lymphocytes in cultures without PHA (10, 15, 16). Unpublished studies in our laboratory have shown that fetal calf
serum also inhibits by 10 to 30% 13H]TdR or [1rs1]UdR incorporation into DNA in cultures of purified lymphocytes,
compared with equal concentrations
of autologous serum
supplementing
the culture medium. The factor or factors in
fetal calf serum that are responsible for this inhibitory effect
are unknown;
it was inconsistently
affected by adsorption of
the sera with hemoglobin-coated
charcoal (unpublished observations). Because the 0.1-mL sample of whole blood also
contains
plasma, the final medium of whole-blood culture
contained approximately
50 mL of autologous plasma per liter,
which presumably
contributed
to cell growth and reactivity.
Prior investigators
niques for karyotyping
tigenic and mitogenic
have used whole-blood
culture tech(17) and measuring
responses
to anstimuli (18-20); these techniques
in-
volve higher concentrations of mitogen, which we find presents
problems
of erythrocyte agglutination.
Our use of less mitogen
avoids agglutination, with results that are reproducible with
a 10% coefficient of variation (triplicate determinations).
Moreover, with minor alterations in the two final steps of our
procedure, one can adapt our method for incorporation
of
other nucleosides.
The
substitution
of [1251]UdR
for [3H]TdR
allows
mea-
surement of the radioactivity in a gamma well counter, which
further simplifies the technique, as shown earlier in dU suppression tests of bone marrow and lymphocyte cultures (3,6).
A maximum pulse time of 3 h at the period of peak response
(day 4), as used in the present study, allows the concentration
of radionucleosides
to be kept relatively uniform throughout
the labeling period and probably avoids internal radiation
damage to the cells during longer incubations (21). Further,
this shorter time obviates the possibility that longer incubation with radionucleosides
might overcome and thereby
conceal dU suppression effects.
76
CLINICAL CHEMISTRY, Voi. 26, No. 1, 1980
of [‘251]UdR or [3H]TdR
was similar to results obtained in cultures
of purified
lymphocytes
from the same
donors in the present studies, and to results in our previously
published studies (3).
dU suppression of incorporation
into DNA in whole-blood culture
The causes of variability in radionucleoside
incorporation
into DNA when fetal calf serum was used in the culture medium are unknown. The role played by the possible presence
of thymidine phosphorylase
(22) in the serum was not investigated. We obviated the problem by not using fetal calf
serum.
Our simple procedure better distinguishes
chronic lymphocytic
leukemia
lymphocytes
from
nonmalignant
lym-
phocytes, with peak incorporation
of isotope being on day 8
instead of day 4 in normal lymphocytes (in the old procedure,
the peak for chronic lymphocytic leukemia cells was on day
4, and on day 3 in normal lymphocytes). In both the new and
the old procedures, the peak of DNA synthesis in malignant
lymphocytes is much lower than in normal lymphocytes.
This “micro
dU suppression”
test should
be of particular
value in accurately diagnosing deficiency of vitamin B12 or
folic acid, or both, because the macro dU suppression test has
been reported to accurately reflect such deficiencies in many
clinical situations in which values for serum vitamin B12 and
(or) folate concentration
are misleading
(3, 6, 23-26).
Supported
initially by private funds, and subsequently in part by
and by USPHS grant no. AM20526.
the Veterans Administration
Note added in proof: Subsequent
studies with N. Colman
suggest that the preferred routine amount of deoxyuridine
for
suppression
tests is less when thymidine
incorporation
in the
control culture (without deoxyuridine)
is low; the preferred
routine amount for whole blood is 2 tmol per culture, for
lymphocytes
10 tmol per culture, and for bone marrow 0.1
zmol per culture. Obviously
these quantities
would change
proportionally
if the ratio of cells to medium is changed.
References
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2(2), 11 (1979).
The dU suppression
test-What
is it? Ligand
CIJNICAL CHEMISTRY, Vol. 26, No. 1, 1980
Q.
77