without excessive shaking of erythrocyte
suspension
(from a
mechanical
shaking bath) is essential for reliable and reproducible insulin-binding
data. Care must also be taken to avoid
hypotonic
shock to which erythrocytes
might be subjected
during incubation
as well as during aliquoting
at the end of
incubation.
We thankfully acknowledge the expert technical assistance of Mary
E. Scott, M.T. (ASCP), and the excellent secretarial assistance of
CLIN. CHEM. 27/4,
609-611
Chiquita P. Mayers in these studies. These investigations were partly
supported by Biomedical Research Support Grant No. 5S07RR05361
from the General Research Branch, Division of Research Sources,
NIH, Bethesda, MD, and partly by the National Foundation March
of Dimes.
References
1. Gambhir, K. K., Archer, J. A., and Carter, L., Insulin radioreceptor
assay for human erythrocytes. Clin. Chem. 23, 1590-1595 (1977).
2. Gambhir, K. K., Archer, J. A., and Bradley, C., Characteristics
of
human erythrocyte insulin receptors. Diabetes 27, 701-708 (1978).
(1981)
Preparation of Whole Blood for Liquid Scintillation Counting
Paul A. Moore
Liquid scintillation
counting of 3H-labeled whole-blood
samples is severely impaired owing to quenching by blood
pigments. In this study, dry oxidation and chemical solubilization followed by decolorization were the two general
methods used to eliminate color quenching. Three blood
volumes were examined: 0.25, 0.50, and 1.0 mL. Dry oxidation yielded complete recoveries
of 3H label with
counting efficiencies
>30% for up to 1.0 mL of blood.
Although blood volumes larger than 0.25 mL can be used
with chemical solubilization and decolorization, treatment
of 0.25 mL of blood gave the highest counting efficiencies,
with count rates comparable to those for 1.0-mL samples.
performance,
use of a compatible
cocktail is imperative
for
proper LSC. Recommended
commercial
procedures
(2, 3)
suggest either acidifying
the cocktail or adding dilute hydrochloric acid after cocktail addition,
to eliminate
chemiluminescence induced by the alkalinesolubiizer.
Acidification
and
incubation
above room temperature
is used, at least in part
to eliminate
chemiluminescence.
In addition
to chemiluminescence
and phase separation,
use of an inappropriate
cocktail
can lead to precipitation
and emulsifier
breakdown.
I have examined various procedures of whole-blood
preparation on the basis of convenience,
counting efficiency,
and
recovery of 3H label.
Materials and Methods
Absorption
and the metabolic
fate of drugs labeled with
tnitium (3H) are often evaluated
by subjecting
a blood sample
to liquid scintillation
counting
(LSC). However,
counting
low-energy
3H particles
in whole blood is difficult, primarily
because of severe color quenching.
Two methods are generally
used to prepare blood samples for LSC: (a) dry oxidation and
(b) chemical
solubilization
followed by decolorization.
Dry
oxidation
provides several advantages
in respect to chemical
treatments:
(a) complete recovery of 3H in the form of 3HOH;
(b) higher counting efficiency; (c) use of larger blood volumes;
(d) separation
of radionuclides
in 3H and ‘4C dual-label
experiments,
and (e) time savings.
Whole blood is usually solubilized
and decolorized
by incubating
it in a commercial
solubilizer
diluted in ethanol or
isopropanol,
then treating it with hydrogen peroxide. The one
advantage
of chemical treatments
with respect to dry oxidation is cost. Commercial
solubilizers, which are toluene-soluble
dimethyldialkyl
quaternary
ammonium
hydroxides
and (or)
chlorides, hydrolyze the blood components
to varying degrees.
A possible alternative
to commercial
solubilizers
is to incorporate a proteolytic
enzyme, trypsin,
into a solubilizing
reagent (1 ).
After solubilization
and decolorization,
an emulsifier-type
LSC “cocktail” is added to prevent phase separation.
Because
commercial
emulsifier-type
cocktails
vary in quality
and
Research Laboratory, Packard Instrument
Co., Inc., 2200 Warrenville Rd., Downers Grove, IL 60515.
Received Nov. 17, 1980; accepted Jan. 19, 1981.
Preparation
of tritiated
blood. Human blood containing
citrate as an anticoagulant,
from a blood bank, was stored at
5 #{176}C.
Nonradioactive
inulin (from dahlia tubers, Sigma) was
used as a carrier for [3H]inulin (dahlia tubers, Amersham)
and
the mixture will be referred to as [3Hjinulin from here on.
[3H]inulmn, 48.6 mg, was thoroughly
mixed with 100 mL of
blood to give an approximate
activity of 0.1 mCi/L. Inulin is
a fructo-polysaccharide
commonly
used for determination
of
renal glomerular
filtration
rate and of extracellular
fluid
volume (4).
LSC conditions.
All samples were counted in quintuplicate
at 15 #{176}C,
each for 2 mm, in a Packard Tri-Carb
460CD with
the 3H channel set at 0 to 20 keV. The Luminescence
Monitor
of the Model 4BOCD (5) was used to detect and correct for
possible chemiluminescence
generated
by solubilization
and
(or) decolorization
of blood.
Sample
preparation
of blood by dry oxidation.
A 306
Sample Oxidizer (equipment
and supplies from Packard) was
used to combust blood samples. The samples were prepared
by placing an aliquot of blood-0.25,
0.5, or 1.0 mL-onto
an
absorbent
Combusto-Pad,
which was inside a CombustoCone. After placing the Combusto-Cone
into the ignition
basket, 0.5 mL of Combustaid
was delivered
onto the Cornbusto-Pad.
Thereafter,
the sample was immediately
burned
for 1.5 mm for 0.25 mL, 2.0 mm for 0.5 mL, and 2.5 mm for 1.0
mL of blood. After automatic
dispensing
of either Monophase-25 or Monophase-40
(18-mL setting), the 22-mL glass
scintillation
vials were capped (plastic liners), and shaken
vigorously
before counting.
CLINICALCHEMISTRY,
Vol. 27, No. 4, 1981
609
Table 1. LSC Sample Preparation Procedures for 0.25 mL of Human Blood Containing [3H]Inulin
Procdurs
St.p B
St.p A
D.colorlzatIonhIncubatlon
no
0.75 mL Soluene-350/lsopropanol
(1/2); 1 hat 40 #{176}C
0.5 mL 30% H202;
0.5 mL 30% H202;
IV
0.5 mL Soluene-350/isopropanol
(1/2); lhat4O#{176}C
0.75 mL Protosol/ethanol (1/2);
1 hat 40#{176}C
0.75 mL Protosol/ethanol (1/2);
V
0.75 mL NCS/isopropanol (1/2);
I
II
III
1 hat40#{176}C
1 hat 40#{176}C
VI
VII
a.
0.75 mL 1% Triton X-100,
1% trypsin, 2% ammonium
bicarbonate; 1 h at 37 #{176}C
1.50 mL Soluene-350/isopropanol
(1/1); 1 hat 40 #{176}C
1/4
h at ambient,a
then 1/2hat4O#{176}C
1/4
h at ambient,
then Y2hat4O#{176}C
0.5 mL 30% H202; ‘/ h at ambient,
then 1/2 h at 40 #{176}C
0.5 mL 30% H202; 1/ h at ambient,
then 1/2 h at 40 #{176}C
0.5 mL 30% H202; 1/4 h at ambient,
then 1/ h at 40 #{176}C
1 mL toluene, then addition of 0.5 mL
30% H202 dropwlse; 1/4 h at
ambient, then /2 h at 37 #{176}C
0.5 mL 30% H202; 1/4 h at ambient,
then 1/2 h at 37 #{176}C
St.p C
Cocktail addItion
15 mL Insta-GeI/0.5
mol/L HCI (9/1)
15 mL lnsta-Gel/O.5
mol/L HCI (9/1)
15 mL Biofluor
10 mL Aquasol 11/
0.5 mol/L HCI (9/1)
15 mL Aq. Comb.
System
10 mL Handifluor
10 mL Handifluor
Sl.p D
N#{149}utrailzatlon
none
none
0.5 mL
0.5 mol/L HCI
none
0.5 mL
0.5 mol/L HCI
none
none
Ambient temperature.
Sample
preparation
of blood by chemical
solubilization
and decolorization.
All of the examined
chemical treatments
are described
in Table 1, and are broken down into three or
four steps: A, B, C, and D. In step A, 0.25 mL, 0.5 mL, or 1.0
mL of blood was added to an aliquot of solubilizer
diluted in
either isopropanol
or ethanol. The mixture was incubated
for
1 h at 40 #{176}C
after swirling the capped vial. The commercial
solubilizers
were: “NCS” (Amersham),
“Protosol”
(New England Nuclear,
NEN), and “Soluene-350”
(Packard).
After
solubilization,
0.5 mL of a 300 g/L solution of hydrogen
peroxide was added in step B, and the loosely capped vial was
gently swirled before allowing it to stand for 15 mm at room
temperature,
followed by incubation
for 30 mm at 40#{176}C.
After
decolorization,
the samples were cooled to ambient
temperature and either a 10- or 15-mL aliquot of LSC cocktail was
added. The vials were capped and vigorously shaken. The LSC
cocktailsused in step C were: “Aquasol II” (NEN)/0.5
mol/L
HC1 (9/1 by vol), “Aqueous Combining
System” (Ainersham),
“Biofluor”
(NEN),
“Handifluor”
(Mallinckrodt),
and
“Insta-Gel”
(Packard)/0.5
mollL HC1 (9/1 by vol). Step D was
an additional
neutralization
step for some procedures
whenever dilute (0.5 molfL) hydrochloric
acid was not premixed
with the cocktail.
Another chemical treatment
recently reported by Michaels
et al. (1), involving a proteolytic
enzyme, trypsin, was also
examined.
Their solubilization
step involved incubating
0.25
mL of blood for 1 hat 37 #{176}C
in 0.75 mL of a mixture containing, per liter, 10 g of Triton X-100 (an emulsifier,
Rohm and
Haas), 20 g of ammonium
bicarbonate
(NH4HCO3,
Fisher
reagent grade), and lOg of trypsin (Bacto 1:250, Difco Laboratories).
Thereafter,
1 mL of toluene
(scintillation
grade,
Mallinckrodt)
was added and followed by dropwise addition
of 0.5 mL of the hydrogen peroxide reagent. The mixture was
allowed to stand at ambient
temperature
for 15 mm before
incubating
for 30 mm at 37 #{176}C.
Once cooled to ambient temperature,
10 mL of Handifluor
was added to the vial, which
was subsequently
capped and shaken.
Treatment
of LSC data. As previously
mentioned,
the
various sample-preparation
procedures
for blood were evaluated based on counting efficiency and recovery of 3H label.
Counting efficiency was determined
by dividing the count rate
(cpm) of the sample by its activity (dprn). A counting
efficiency correlation
curve was generated
by external
standardization.
The recovery of 3H was found by dividing the
average activity (dpm/volume
of blood) of the noncombusted
610
CLINICALCHEMISTRY,Vol.
27, No. 4, 1981
sample by the average
covery by combustion
activity of the combusted
was taken as 100%.
sample.
Re-
Results and Discussion
As expected,
dry oxidation
provides the most convenient
means of blood preparation
with the highest counting
efficiency. Counting efficiency averaged 32.0% for 1.0 mL of blood
with Monophase-40.
Memory and 3H spillover
into the ‘4C
section were found to be approximately
equivalent
to background. Not only does dry oxidation yield complete analytical
recoveries,
it eliminates
chemiluminescence,
as indicated
by
the 460CD Luminescence
Monitor.
Because dry oxidation
eliminates color quenching,
the counting efficiencies, and thus
activity, can be determined
without laborious
internal standardization,
which is necessary
when color quenching
is severe.
Although
0.5 and 1.0 mL of blood were used, only preparation of 0.25 mL of blood by two chemical procedures
produced reproducible,
clear, colorless, homogeneous
LSC samples. These two chemical procedures
as described
in Table 1
were: (land II) Soluene-350/isopropanol
(1/2) with acidified
Insta-Gel
(2) and (IV) Protosol/ethanol
(1/2) with acidified
Aquasol 11(6). Elimination
of the severe color quenching
by
these procedures
used on 0.25 mL of blood not only provides
higher counting efficiencies,
but eliminates
the need for determining
counting
efficiencies
by time-consuming
internal
standardization.
In addition,
the relatively high counting efficiency (nearly 30%) with 0.25 mL of blood allows for count
rates that are comparable
to or better than achieved for 0.5
or 1.0 mL of blood. Based on these findings, preparation
of
0.25 mL of blood is recommended
for achieving reproducible,
clear, colorless,
homogeneous
LSC samples
with relatively
high counting
efficiencies
and count rates. If larger blood
volumes are necessary,
the dry oxidation
is the method of
choice.
As shown in Table 2, procedures
I (Soluene-350/isopropanol
(1/2) with acidified Insta-Gel)
and IV (Protosol/ethanol
with
acidified Aquasol II) gave comparable
counting
efficiencies
(28.0% and 28.1%, respectively).
In addition,
both methods
yielded high calculated
recoveries
of 3H label: 101.9% and
91.2% for procedures
I and IV, respectively.
By substituting
Aquasol II for Biofluor in the prescribed
NEN procedure
(3), precipitation
was avoided. A reduction
in the incubation
temperature
to 40 #{176}C
from 60 #{176}C
as described in the NEN procedure
(3) could possibly
have ac-
Table 2. Average Counting Efficiencies, Average Activities, and Recoveries of 3H for Various LSC
Sample-Preparation Procedures for 0.25 mL of Human Blood Containing 3H-inulln
Procedure
Procedure’s
no.
cocktaIl
I
II
III
IV
V
VI
VII
VIII
solublllzer.
system
Appearance
0.75 mL SoIuene350/IPAa
homogeneous, clear,
15 mL acidified Insta-Gel
colorless
homogeneous, clear,
0.5 mL Soluene-350/IPA
15 mL acidified Insta-Gel
colorless
precipitation
0.75 mL Protosol/ethanol
15 mL Biofluor
homogeneous, clear,
0.75 Protosol/ethanol
10 mL acidified Aquasol II
colorless
0.75 mL NCS/IPA
phase separation
15 mL ACS
0.75 mL 1% Triton X-100,
homogeneous, clear,
1% Trypsin, 2% NH4HCO3 yellow
10 mL Handifluor
1.50 mL Soluene-350/IPA
homogeneous, clear,
10 mL Handifluor
yellowish-green
Combustion in 306 Sample
homogeneous, clear,
Oxidizer using 18-mL
colorless
setting for Monophase-25
No.
Av countIng
efficiency
samples
(% ± SD)
Av
(dpm
acty
± SD)
R.covery,
5
28.0
0.3
62263 ± 2521
101.9
5
30.5 ± 0.3
48481 ± 2392
79.4
5
%
inconcl usive due to precipitation
5
28.1 ± 0.4
55697 ± 592
91.2
5
26.3±1.1
41343±1014
67.7
5
21.1±0.6
46271±812
75.7
68.6
5
1.1±0.2
41911±6788
5
42.8±0.8
61089±5176
100
#{149}
IPA: Isopropanol.
counted
for the precipitation
when Biofluor was used. Precipitation
can cause severe quenching
in 3H labeled samples
in the form of sample self-absorption,
where the low-energy
beta electrons are not in intimate proximity to primary solvent
molecules of the LSC cocktail. The suggested
10-mL volume
of Aquasol 11(6) used at the same incubation
temperature
(40
#{176}C)
did not result in precipitation
with 0.25 mL of blood.
The procedure
using NCS tissue solubilizer/isopropanol
(1/2) with Aqueous
Combining
System cocktail resulted
in
phase separation
giving a recovery of about 68% as shown in
Table 2.
As also apparent
from Table 2, the enzymatic
solubilizer
procedure
(1) resulted
in a counting
efficiency
of 2 1.1%,
comparable
to that reported by Michaels et al. (1) for 0.2-0.3
mL of blood. Analytical
recovery for 0.25 mL of blood was
incomplete
(75.7%) in this study, in contrast with the complete
recovery previously
reported
(1). This apparent discrepancy
may be explained
by the difference
in the chemical properties
of the 3H-labeled
material.
A tissue solubilizer
has been reported (7) to enhance recovery of aqueous 3H-labeled
inulin.
Blood sample preparation
without the use of a quaternary
ammonium
hydroxide
solubilizer
may possibly be limited to
noninulin or other non-plating
tritiated samples. The resulting
yellow color in the LSC samples apparently
accounted
for the
comparatively
low counting
efficiency
of this method.
Logistically,
the prolonged
dropwise addition
of 0.5 mL of hydrogen peroxide reagent into the solubilizer
blood mixture is
time-consuming.
Prior addition
of 1 mL of toluene did not
eliminate
excessive foaming when the H202 was not added
dropwise. Each vial required about 3 to 5 mm for completion
of H202 addition.
This chemical procedure
was found inappropriate
for larger blood volumes, 0.5 and 1.0 mL, due to
severe color quenching.
Due to the aqueous nature of blood and the composition
of
tissue solubilizers,
an appropriate
emulsifier-type
cocktail is
required.
To illustrate
how an inappropriate
cocktail can affect the counting efficiency of a sample the use of Soluene-350
with Handifluor
was repeated
as reported
by Michaels et al.
(1). This procedure, as expected, yielded a low counting efficiency, 1.1%, as shown in Table 2. However, use of the current
and recommended
procedure
(2) of Soluene-350/isopropanol
(0.5 mL, 1/2) with Insta-Gel
(II) as described
in Table 1
yielded clear and colorless homogeneous
LSC samples with
a counting efficiency of 30.0%, as appears in Table 2.
I conclude that combustion
is the recommended
method
for preparing
blood volumes greater than 0.25 mL. If dry oxidation cannot be used, then the best alternative
procedure
found in this study is to solubilize 0.25 mL of blood with 0.75
mL of Soluene-350/isopropanol
or Protosol/ethanol
(1/2) for
1 h at 40 #{176}C,
followed by decolorization
with 0.5 mL of hydrogen peroxide
reagent and, finally, addition
of 15 mL of
Insta-Gel
or Aquasol 11/0.5 mol/L HC1 (9/1).
References
1. Michaels, B., Hahn, E., and Kenyon, A., An improved procedure
for solubilization and assay of blood and feces in liquid scintillation
counting. Anal. Biochem. 99, 288 (1979).
2. Anon., Insta-Gel Technical Bulletin, Packard Instrument Co.,
Downers Grove, IL, 1978.
3. Kobayashi, Y., and Harris, W., LSC Application Notes, no.2, New
England Nuclear, Boston, MA, 1978.
4. Koushanpour,
E., Renal Physiology: Principles and Functions,
W. B. Saunders Co., Philadelphia, PA, 1976, pp 20,96.
5. Anon., Tri-Carb 460C and 460CD Liquid Scintillation
System
Operation Manual, Packard Instrument Co., Downers Grove, IL,
1979.
6. Anon., Aquasol II Technical Bulletin, New England Nuclear,
Boston, MA, 1978.
7. Moore, P., Improved liquid scintillation counting of tritiated inulin.
Clin. Chem. 27, 349-350 (1981). Letter.
CLINICAL CHEMISTRY, Vol. 27, No. 4, 1981
611