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Theestimationofbloodvolumeisimportantinstudies
of radiopharmaceutical distribution. In spite of this,
values used by various investigators differ enough to have
a significant impact on reported results. The measure
ment of plasma volume (1—4)and of RBC volume (5—8)
have been studied, but there are few reports on simul
taneous measurements of plasma volume and RBC
volume (9—12)for blood-volume estimation.
Estimates of blood volume in the rat derived from the
plasma dilution method using T-l824 and 1-131 human
serum albumin vary from ‘-@‘4.3to 8.0 ml per 100 g BW, with a mean of'-.-7.0 ml. Blood volumes calculated on the
basis of the dilution of labeled RBCs range from 4.5 to
6.3 ml per 100 g BW, with a mean of 5.7 ml. The errors
encountered in making blood-volume determinations by
dilution of either plasma or red cells alone have been
pointed out by many investigators (12—15).
Although there are a few reports of the simultaneous
measurement of plasma and RBC volumes for deter
mination of total blood volume, the change in the nela
tionship between blood volume and body weight as body
Received Feb. 8, 1984; revision accepted Sept. I 2, 1984. For reprintscontact: M. DonaldBlaufox,MD, PhD, Dept.of Nu clear Medicine,Atbert Einstein Cottegeof Medicine, 1300 Morris Park Ave., Bronx,NY 10461.
weight increases has not been considered previously.
Younger rats have a larger blood volume relative to their
body weight than older rats (4,6). Many studies are
carried out during periods of rapidly changing body
weight, which emphasizes the importance of these re
lationships.
The present study was undertaken to determine the
relationship between blood volume and body weight,
using RBCs labeled with Tc-99m for RBC volume, and
human serum albumin labeled with 1-125 for plasma
volume. These results yield the total exchangeable blood
volume (TBV).
METHODS
Twenty-nine female and 41 male Wistar rats,
(99.5—414g), fed Purina Laboratory food and water ad
libitum, were studied. Experiments were standardized with respect to time of day to avoid diurnal changes in
blood volume (12).
The femoral (or carotid) artery and femoral vein were
cannulated under light ether anesthesia using silastic
tubing (medical grade, 0.012 i.d., 0.025 o.d., 10 in. long).
The cannulae were filled with 20 units of hepaninized
saline and the free end brought through the tail. The rats
were allowed to recover for an hour before study.
72 LeeandBlaufox^ The Journal of Nuclear Medicine
Blood Volume in the Rat
H. B. Lee and M. D. Blaufox
Department ofNuclear Medicine, Albert Einstein College ofMedicine, Montefiore Medical Center, Bronx, New York
The organ distribution of radiopharmaceuticals in the rat is usually estimated using 7%
of body weight (BW) for blood volume (By). In spite of its important Impact on the
evaluation of new agents, this value has not been validated adequately.We therefore
studied blood volume in 70 awake Wistar rats (100 to 400 g BW) in which red blood
cell volume (RBCV)and plasma volume (PV)were measuredsimultaneously.Red
bloodcellvolumewasmeasuredbyinvitroABC-taggingwithTc-99minSn
pyrophosphate, 0.05 @gper ml of blood; plasma volume was measuredwith 1-
human serum albumin (HSA).Ten minutes after injection of the dose, 0.5 ml of blood
was withdrawn from the carotid or femoral artery and duplicate samples of 0.025 ml of
blood were counted after separating ABCs from plasma. Total blood volume was
calculatedbyaddingABCvolumeandplasmavolume.Therelationshipfor theentire
group was: BV (ml) 0.06 X BW + 0.77 (r 0.99, n 70, p <0.001). The difference
between male and female rats was not statistically significant. The use of an arbitrary
value of 7% for estimation of blood volume can lead to significant errors in calculating
radiopharmaceuticaldistribution.Theuseof thegeneralformulafor the blood-volume
calculation described here should improve the accuracy and reliability of estimates of
radiopharmaceutical distribution.
J NuciMed25:72—76, 1985
BWgBV1(ml/lOOg)n
0.50222 BW< 120(g) 114.22±8.7440.00 ±2.464.68 ±0.572.12 ±0.176.80 ± 0.40483 BW > 120 (g) 270.19 ±80.4043.94 ±2.773.92 ±0.322.27 ±0.176.19 ±
0.39294 Female 175.4 ±46.8643.28 ±2.984.06 ±0.412.26 ±0.26.33 ±
0.60415 Male 253.5±112.642.30 ±3.394.22 ±0.642.22 ±0.196.44 ±
0.5270* Total 221.18±98.742.70 ±3.244.16 ±0.542.24 ±0.196.40 ±
weight.t BW body hematocrit.t HCT volume.§ PV plasma volume.IRBCV = red-blood-cell BVRBCV.@±s.d. PV +
catheter was removed at the end of the study and
counted. Any tracer left in the catheter was subtracted
from the injected dose counts.
Five and ten minutes after 1-125 HSA and Tc-99m
RBC blood injections, @‘0.06ml of arterial blood was
obtained. Using a micropipet, 0.025 ml of blood was
transferred into another capillary tube. Its tip was sealed
and the tube centrifuged for 4 mm.
The hematocnit was calculated and the packed RBC
in the capillary tube were carefully separated from
plasma and placed in a 5-mi counting tube. Samples were
counted in a well scintillation counter with a 20%window
peaked at 140 keY. After 3 days (for Tc-99m decay),
samples were counted again for 2 mm for 1-125 (20%
window centered on 35 keV).
CALCULATIONS
RBC volume was obtained by dividing the cpm of the
total injected Tc-99m RBC dose by the cpm of RBC
tracer. The mean of the 5- and 10-mm samples was
used.
RBCV = cpm of Tc-99m RBC injected dose
cpm/ml of Tc-99m RBC
Estimated BV by RBCV = RBCV X 100,
Hct X CF
where CF = hematocnit correction factor (0.96);
cpm of I 125 HSA injected dose
and PV =
cpm/ml of plasma
. PVx Estimated BV by PV =
(100 —Hct)
The estimated blood volume was calculated from
plasma volume without considering the plasma trapped
in the packed ned cells after centnifugation. The true
blood volume was calculated by adding RBC volume and
plasma volume. Statistical analysis was performed by
group t-test and one-way analysis of variance.
PLASMA VOLUME DETERMINATION
Fifty or 100 s1 of 1-125 human serum albumin
(0.5—0.1zCi) was injected through the femoral-vein
catheter. The actual injected dose was calculated from
the change in weight of the dose syringe. A standard was
prepared by diluting 0.025 ml of I- 125 HSA with 10 ml
of distilled water. The 0.025 ml of diluted standard was
used for the dose calculation. Blood samples of 0.05 ml
were drawn from the femonalor carotid artery S mm and
10 mm later, placed in two heparinized capillary tubes,
0.025 ml each, and analyzed to determine th hematocnit
(Hct). Femonal arterial samples were drawn from larger
rats and carotid samples from smaller rats.
RED-BLOOD-CELL VOLUME
DETERMINATION
Half a ml of blood was withdrawn from the femoral
artery and placed in a hepaninized test tube. The blood
was incubated for 10 mm at room temperature with 0.
ml of normal saline, containing 0.05 sg of stannous py
nophosphate (Sn-PPi), per ml of blood. After incubation,
10 @Ci99mTcO4in 0.01 ml of normal saline was added
and incubated for 10 mm. Following incubation period,
25 jsl of the blood labeled with Tc-99m RBCs was pi
petted into two heparinized capillary tubes (1.2 mm i.d.,
1.4 mm o.d., 75 mm long) for the dose calculations. The remaining Tc-99m-RBC blood was weighed before in
jection. The RBC tagging method is a modification of
the technique described by Korubin (16). Ninety-five
percent of the Tc-99m is on the RBCs at time zero and
the counts/ml RBC do not change significantly up to #@.s3Ømm, when elution of the label becomes detectable.
The blood radioactivity is 96.5% on the RBC at S mm
and 97.2% at 10 mm. A known volume of the Tc-99m-
RBC blood was injected through the femoral-vein
catheter immediately after I-i 25 HSA injection, using a tuberculin syringe and a 27-gauge needle. The catheter was flushed with 0.02 ml of normal saline. The femonal
TABLE I
Body Weight and BloodVolumes In Wistar Rats HCTt (%) PV* (ml/100 9) RBCV@(ml/100 9)
Volume 26 •Number 1 •January 1985 (^73)
n BW(9) HctMethod
used for RBCVMethod
used for PVPV
(ml)per
100g BWBV
(ml)per
BWRef.BV 100g
fromPV(ml) 12 189.0 40. 12 53.4 37. 9 81.2' 35. 13 197.9' 43.
43 241.0 46.11-
HSA
CO
CO
CO
T-
l-125HSA5.
3BV
(ml)12 fromRBCV
196.3 47. 31 231.7 45. — <100.0 — — >100.0 — 34 239.0 — 16 326.0' 48.4P
P
Fe- Fe- P Fe-552.
— — — 2.34.
8BV
fromRBCV+ PV(ml)
11 254.0 50. 35 393.0 46. 1OM 180—250 36. 18F 180—250 36. —M 101—125 42. —M 226—250 48.6P
P
Fe- Fe- Fe- Fe-592.
2.07T-
T-
l-125HSA 1-131HSA T- T-18244.
= mean.
TABLE 2
Previous Studies of Blood Volumes RBCV (ml)per 1009BW
may be explained in part by the weight of animals used
and differences in methods. Several studies of blood
volume in rats by other investigators are listed in Table
- In general, the mean blood volume per 100 g body
weight is lower than the value reported here. Garcia (6),
Lippman (4), and Belcher (19) reported that animals
weighing <100 g have relatively larger blood volume.
The present study confirms that the group of rats with
mean BW 114 ±8.7 have higher blood volume than the
group with mean BW 270 ±80 g (p <0.0005). The fe
male rat weight reported here did not exceed 250 g be
cause virgin rats rarely exceed this weight.
The accurate estimation of blood volume is critical to
the performance of radiopharmaceutical distribution
studies. These data demonstrate that the generally ac
cepted figure of 7% overestimates the blood volume in
most animals. Such an overestimate would lead to errors
in determination of organ distribution and blood back
ground estimates. The significant differences in relative
blood volume in animals of different size are important
in reconciling results of different investigators.
The linear regression described here appears to be
reliable for estimating rat blood volume, and is necom
mended for general use in radiophanmaceutical distni
bution studies in the rat.
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76 LeeandBlaufox The Journal of Nuclear Medicine
