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Determining Blood Volume in Rats: A Comparison of Plasma and Red Blood Cell Volume, Slides of Nuclear medicine

A study conducted by H.B. Lee and M.D. Blaufox to determine the relationship between blood volume and body weight in rats. The researchers used Tc-99m and 1-125 human serum albumin to measure RBC volume and plasma volume simultaneously. The study found that the estimated blood volume from RBC volume and plasma volume were significantly different, and the accurate estimation of blood volume is critical for radiopharmaceutical distribution studies.

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Theestimationofbloodvolumeisimportantinstudies
of radiopharmaceutical distribution. In spite of this,
values used by various investigators differ enough tohave
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.
The Journal of Nuclear Medicine
72 LeeandBlaufox
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 Impactonthe
evaluation of new agents, this value has not been validatedadequately. 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 measured simultaneously.Red
bloodcellvolumewasmeasuredbyinvitroABC-taggingwithTc-99minSn
pyrophosphate, 0.05 @gper ml of blood; plasma volume was measured with 1-125
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.Therelationshipfortheentire
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 the generalformulafor theblood-volume
calculation described here should improve the accuracy and reliability of estimates of
radiopharmaceutical distribution.
J NuciMed25:72—76,1985
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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

  1. 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.

REFERENCES

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plasma volume partitions in the growing rat. Am J

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The plasma volumeof the Wistar rat in relation to the

body weight. Experientia 37:381—382, 1981

  1. Lippman RW: Blood, plasma, and “drawnblood―vol umes intherat.ProcSoc Exp BiolMed 66:188-191, 1947
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volumeof the adult rat, as determined by Fe59and P

labelledred cells.Proc Soc Exp Biol Med 71:176—178,

6. GarciaiF: Changesin blood,plasmaand redcellvolume

in the male rat, as a function of age. Am J Physiol 190: 19—24, 1957

  1. Montgomery P O'B: A method for determining blood

volumeof the rat usingradioactivephosphorus.ProcSoc

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cellvolumeof the adult rat as measuredby taggedcells.

ProcSocExp BiolMed 74:681-685, 1950

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  1. Wang L: Plasma volume, cell volume, total blood volume

and F cells factor in the normal and splenectomized

Sherman rat. Am JPhysiol 196:188—192, 1959

  1. Huang KC, Bondurant iM: Simultaneous estimation of plasma volume, red cell volume and thiocyanate space in unanesthetized normal and splenectornized rats. Am iPhysiol185:441-445, 1956
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blood (Fe59)and plasma (1131)volumesof rats deter

mined by liquid nitrogen freezing. Circ Res 4:419-424, 1956 I3. Reeve EB, Gregersen MI, Allen TH, et at: Distribution

ofceltsand plasmain the normaland splenectomizeddog

and its influencein bloodvolumeestimateswith P32and

1-1824.Am J Physiol175:195—203, 1953

  1. Griffith iQ, Campbell R: A method for determining

bloodvolumein rats. ProcSoc Exp BiolMed 36:38-40,

  1. Gregersen MI: Blood volume. Ann Rev Physiol 13: 397—412, 1951

16. Korubin V, Maisey MN, McIntyre PA: Evaluationof

technetium-labeled red cells for determination of red cell

volumein man. J Nucl Med 13:760-762, 1972

  1. Leeson D, Reeve EB: The plasma in the packed cell

column of the haematocrit. J Physiol 115:129-142,

I

  1. Owen CA, Power MH: Intercellular plasma of centni

fuged human erythrocytes as measured by means of

iodo'31-albumin.J ApplPhysiol5:323-329, 1953

19. BetcherEH, HarnissEB: Studiesof plasmavolume,red

cell volume and total blood volume in young growing rats. JPhysiol139:64—78, 1957

76 LeeandBlaufox The Journal of Nuclear Medicine