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Perspectives on Animal Use

Michael W. McGuill and Andrew N. Rowan


INTRODUCTION
The purpose of this paper is to review the literature
on the effects of acute and chronic blood loss and
techniques of blood sampling in laboratory animals.
When possible, recommendations regarding limits on
single and multiple blood samplings in laboratory
animals are offered.
Although blood sampling is a common laboratory
procedure, it is nonetheless problematic, especially
for techniques that attempt to set up protocols for
chronic blood taking. In a letter to The American
Journal of Physiology, Giner et al. (1987) announced
their failure to reproduce an experimental protocol on
rats that involved repeated blood sampling by means
of an arterial catheter described in an earlier article in
the same publication (Burt et al., 1980). Giner and
associates attempted the described procedure on eight
rats with no success. They then modified the technique
by increasing the heparin concentrations, by using
larger catheters, by flushing the cannula frequently,
and finally by using a Teflon cannula. They succeeded
in obtaining blood samples, but not without causing
renal or intestinal infarctions and ischemia to the hind
limbs. According to the authors, "after three months
of work in attempting to emulate Burt's work and the
death of a large number of rats, we were left with an
excellent model for arterial embolism."
Giner's letter highlights a problem basic to many
vascular sampling techniques: their successful execution requires considerable skill. The difference between one experimenter's success with a technique
Michael W. McGuill is a third-year veterinary student at Tufts
University School of Medicine. Andrew N. Rowan is associate
professor in the Department of Environmental Studies and director
of the Tufts Center for Animals and Public Policy at the Tufts
University School of Veterinary Medicine. He is a member of the
ILAR News Editorial Panel and the ILAR Committee on Pain and
Distress in Laboratory Animals.
Volume 31, Number 4

Fall 1989

and another's failure may have as much to do with


differences in the experimenter's technical skill as with
any flaw inherent in the technique.
The questionable reproducibility of the many techniques, for whatever reasons, is not the only problem
facing the researcher making decisions about blood
sampling. Another problem concerns the amount of
blood that can be withdrawn from laboratory animals
without causing injury or stress, either in a single
sampling or in multiple samplings. Most facilities have
their own rules of thumb. For example, many animal
care and use committees recommend a limit of
1 percent of the total body weight for a single sampling.
However, such guidelines may be based more on
custom than on scientific studies describing blood
volume losses that cause minor to significant physiological and psychological stress.
The issue is complicated further by the problems of
judging an animal's psychophysiological state. Analogies from human experience may be of limited help.
For instance, after a loss of 15-20 percent of the total
blood volume, a man or woman will probably feel
nausea, experience dizziness, have blurred vision, and
is likely to faint. However, laboratory animals have
not been reported to lose consciousness after a blood
loss of 15-20 percent. Nevertheless, laboratory animals
may conceivably experience some symptoms of the
vasovagal response, such as nausea. At this point we
do not know.
EFFECTS OF BLOOD LOSS AND BLOOD
SAMPLING O N RESEARCH DATA

In this section we look at a number of experimental


variables associated with blood sampling and blood
loss. Each of the variables has the potential simultaneously to affect research data and to cause distress
in research animals (Rose, 1987).

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Biological Effects of Blood Loss:


Implications for Sampling Volumes and Techniques

Overview of Physiological Responses to


Hemorrhage

Manipulation and Acclimatization

Not only is the trauma of blood loss stressful to the


animal, but manipulation alone is stressful. Handling
for as little as five seconds has been shown to cause
significant increases in corticosterone levels (Seggie
and Brown, 1975). Besch and Chou (1971) reported
that decreases in plasma glucose levels are directly
related to handling time. (Plasma glucose changes
reflect stress because stress-released epinephrine raises
blood sugar by its hyperglycemic action on the liver.)
Mattheij and van Pijkeren (1977) observed that simply
handling rats and placing them in an empty jar for 45
seconds or transferring them to another cage in the
same room induced significant increases in prolactin
levels.
Few studies specifically contrast stress levels of
tamed, acclimated animals undergoing blood sampling
with those of untamed, unacclimated animals. However, several studies have described how stress experienced by untamed or unacclimated animals confounds experimental results. For example, responses
to toxins are adversely affected if the animals are not
acclimated to experimental conditions (Damon et al.,
1986); likewise, animals not tamed by handling react
to euthanasia with significant changes in blood values
that are not seen in tamed animals (Uphouse et al.,
1982). Researchers who wish to reduce the stress of
blood sampling, therefore, will need to habituate the
animals to gentle handling and must reduce handling
time as much as possible. Furthermore, they must
consider the time required for various sampling techniques as an important element in deciding which is
least stressful.
Puncture

All blood-sampling techniques are invasive. Vessel


puncture, appendage amputation (tail transection, toe
clipping), or vessel incision all presumably cause at
least some pain and stress if used without anesthesia.
One study of humans suggests that stress caused by
venipuncture accompanied by minor blood volume
sampling is not sufficient to change prolactin levels,
at least in pregnant women (Ferriani and De sa Silva,
1985). However, in a study on rhesus monkeys by
Heindon et al. (1984), stresses associated with capture
and venipuncture were shown to increase levels of
growth hormone (GH) and cortisol, but not testosterone or prolactin. Another study, by Calligaris and
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With minor blood losses, animals may be asymptomatic. With losses of approximately 10 percent or
less of the total blood volume, baroreceptor-initiated
reflexes cause release of cholinergics from the adrenal
medulla and sympathetic nerve endings. As a result,
(1) heart rate increases, (2) arteriolar beds in muscle
and skin constrict, and (3) veins and venous reservoirs
constrict. Thus arterial pressure, venous return, and
cardiac output are maintained or minimally affected.
Slower-acting compensatory mechanisms that help
replace lost volume include secretion of antidiuretic
hormone and activation of the renin-angiotensin aldosterone system.
With moderate blood losses the animal will suffer
drops in arterial pressure and cardiac output despite
compensatory mechanisms. Losses of approximately
15-20 percent of the total blood volume cause massive
cholinergic release with tachycardia and intense arteriolar constriction with further redistribution of blood
away from the gut and skin. Venous constriction
partially sustains venous return, and mobilization of
interstitial fluid to the intravascular compartment over
time restores some of the lost fluid volume. Anaerobic
glycolysis occurs due to the lack of oxygen. The
increased plasma lactate causes metabolic acidosis,
and a compensatory tachypnea ensues.
With further blood losses, drops in cardiac output,
blood pressure, and tissue perfusion become life threatening. Tissue anoxia, hypercapnia, and acidosis can
lead to widespread cell injury and irreversible tissue
damage, organ compromise, and death. With severe
blood losses, cardiac function is limited by decreased
cardiac perfusion as well as myocardial depressant
factor, which is released by the poorly perfused pancreas. Late in shock, decreased perfusion of the
medullary vasomotor center causes diminished compensatory reflexes.
Researchers should plan and execute each sampling
protocol with an appreciation for the stresses associated with blood loss to the animal and do whatever
they can to minimize the animal's reaction to the
stress. Careful planning and control of blood sampling
and all experimental variables associated with it should
not only improve the state of the animal, but also
minimize confounding influences on research data.
Dodds (1987) has four suggestions to researchers
wishing to minimize changes in blood components
while sampling blood: (1) select a more tractable
species or calm individuals, (2) precondition the animals to adapt them to handlers and procedures, (3)
use a sampling technique that minimizes changes in
blood values, and (4) note and control the use of
anesthetics, because these may change blood values.

Because all of these suggestions are associated with


stress reduction, following them will benefit the animal
as well as the scientific results.

Taleisnik (1983), used heart puncture by itself as a


stressor to measure prolactin release under the influence of variables such as time of day.
Anesthesia

Catheter

Sampling blood through an indwelling catheter is


generally considered to be less stressful and to cause
fewer changes in blood variables than sampling with
a needle and syringe (Flynn and Guilloud, 1988).
Catheter sampling is particularly useful in studies
requiring multiple blood collections. However, some
research indicates that indwelling catheters can affect
basal levels of hormones. For instance, Fagin et al.
(1983) reported that individually housed rats with
external carotid artery cannulas had slightly elevated
morning levels of plasma adrenocorticotropic hormone
and corticosterone. After intravenous (IV) injection of
saline in rats via femoral artery or vein cannulas,
Lestage et al. (1985) found that plasma corticosterone
levels were 2.4-fold lower than in restrained rats, but
levels still indicated moderate stress.
Richman et al. (1980) noted that platelet counts fell
64 percent in dogs after 48 hours of catheterization
with Swan-Ganz pulmonary artery catheters.
Volume 31, Number 4

Fall 1989

With a slower rate of sampling, a greater volume


can be removed without stressing the animal. In fact,
Jain (1986) reported that a 50-percent blood volume
loss, if slow, will not be accompanied by clinical signs
of shock. Mattheij and van Pijkeren (1977) observed
that a loss of up to 3.0 ml withdrawn over five hours
from rats weighing at least 400 g, or roughly 13 percent
of the total blood volume, did not cause a change in
serum prolactin.
BIOLOGICAL EFFECTS OF A SINGLE
HEMORRHAGE
Problems in Estimating Blood Volumes and
Losses

We recommend estimating blood volumes based on


body weight to determine guidelines for blood-sampling
volumes in research animals (Table 1). This is obviously more practical than measuring the blood volume of each individual animal, but there are limits to
making such estimates. For instance, a blood volume
estimate for a single species may not reflect differences
among individual breeds or variations due to age, size,
or illness. According to standard texts, estimates of
blood volume based on surface area or lean body mass
tend to be closer to actual determined volumes, but
are impractical for standard laboratory purposes.
Our discussions of the literature report the percentage of total blood removed from an animal when the
data have been provided in the original source. If such
data have not been provided, we have estimated
percentage of blood volume removed based on body
weight. However, sometimes only absolute volumes
of blood removed are reported. In such cases it is
impossible to know what percentage of total blood
TABLE 1 Average Blood Volumes of Research Animals Based
on Body Weight

Species

Whole Blood
Volume
(ml/kg)

%
Body
Weight

Dog

86

8.6

Cat

56 (47-66)
67"
43*.

6.7"
4.3*

Plasma
Volume
(ml/kg)

Approximate Absolute
Blood Volumes of Animals
of Described Weight

50

14-kg (30 Ib) dog: 1200 ml

41 (35-52)
48"

4.5-kg( 10 1b) cat: 252 ml


4.5-kg (10 1b) cat: 300 ml"
4.5-kg(10lb)cat: 194 ml''

Rat

64 (58-70)

6.4

40 (36-45)

300-g rat: 20 ml

Guinea pig

75 (67-92)

7.5

40 (35-48)

90-g guinea pig: 68 ml

Rabbit

56 (44-70)

5.5

39(28-51)

3.2-kg rabbit: 180 ml

NOTE: All values from Altman and Dittmer (1974) unless otherwise specified.
"Jain (1986).
''Breznock and Strack (1982).

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Often, anesthesia can increase the stress of a sampling procedure. Wiersma reported that taking blood
samples from an atrial cannula in the rat was more
stressful when light ether was used because prolactin
levels were significantly elevated only when ether was
used (Wiersma and Kastelijn, 1985). Mattheij and van
Pijkeren (1977) observed an increase in prolactin levels
after a 45-second ether stress.
Furthermore, some anesthesias cause more stress
than others. Rats subjected to a 2.5-2.7 ml/100 g
hemorrhage over 10 minutes had a much lower survival
rate when anesthetized with intramuscular sodium
pentobarbital than with inhalant methoxyflurane (Yale
and Torhorst, 1972). Upton and Morgan (1975) found
little difference in blood parameters from the effects
of ether, pentobarbitone sodium, and fentanyl-droperidol while obtaining cardiac blood, although the use
of manual restraint significantly increased blood acidity
and raised the levels of hemoglobin, hematocrit, and
plasma protein, presumably because of stress. Lawson
and Gala (1974) observed prolonged increases in plasma
prolactin following ether and intraperitoneal injection
of sodium pentobarbitone, but no change in plasma
prolactin following ketamine or intraarterial sodium
pentobarbitone. (Refer also to "Mortality" on page 13
for references to studies on anesthesia effects in pigs.)

Rate of Bleeding

sizes. Larger rats had lower percentage blood volumes


than smaller rats (Table 2). The mortality rates for
different sized rats are given in Table 3.
Researchers should keep in mind that many variables
in addition to loss of blood volume affect an animal's
response to blood sampling. These include the rate of
blood loss, the site and technique of withdrawal, the
skill of the bleeder, the use and type of anesthesia,
the age and sex of the animal, and its nutritional and
health status. To give one example, if an animal is
obese, the researcher should lower an estimate of
blood volume and base it on the animal's expected
normal weight.
There is obviously great potential for error in determining blood volumes, which leads to serious discrepancies in the literature. These are further reasons
why investigators should be cautious when determining
limits on blood-sampling volumes based on estimates
from standardized tables. When possible, the investigator should use the lower, more prudent limits.

Massive Blood Loss


Sustained Hypotension

A frequently studied model of hemorrhagic shock


has been the standardized Wiggers model of hemorrhage (Wiggers, 1950). In this model, the anesthetized

TABLE 2 Relationship Between Body Weight and Blood


Volume in Rats
Body Weight (g)

Total Blood
Volume (ml)

% Body Weight
(ml/100 g body weight)

250

17.4

6.97

300

19.5

6.51

350

21.1

6.04

400

22.3

5.58

SOURCE: Satoet al. (1985).

TABLE 3 Relationship Between Body Weight, Mortality, and


Blood Volume Loss in Rats

Body Weight (g)

Total Blood Volume


Removed (ml) per
100 g Body Weight,
LD 50

250

2.7

39

300

2.6

40

350

2.5

42

400

2.4

43

% Total Blood
Volume Removed.
LD50

SOURCE: Sato et al. (1985).


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volume was removed from an animal. Also, most of


the data we found were obtained from studies on rats
and dogs. There were very few data on effects of blood
loss in the rabbit or mouse.
Significant differences in blood volumes exist between animals of the same species. Courtice (1943),
using the Evans-blue dye method, observed the mean
total blood volume of a group of mongrels to be
79 ml/kg and the mean total blood volume of a group
of greyhounds to be 114 ml/kg. This represents a
35 ml/kg difference.
A hypothetical example based on these observations
demonstrates the need to err on the side of caution
when determining limits of nonstressful blood-sampling
volumes. If, for instance, researchers working with
mongrels had records only of the total blood volume
of greyhounds and thought they were removing
20 percent of blood volume from the mongrels based
on the greyhound data, they would actually be removing 22.8 ml/kg, or about 30 percent, of the mongrels'
blood volume.
Discrepancies in the literature on the average blood
volumes of animals are common. One reason may
involve the technique used to measure blood volume.
The International Committee on Standardization in
Haematology recommends that investigators determine blood volume by measuring red cell volume using
sodium radiochromate or sodium pertechtate as a red
cell label and measuring plasma volume using human
serum labeled with radioiodine as a plasma label (Jain,
1986). According to one researcher, the Evans-blue
dye (T-1824) method, the most common of the early
methods, overestimates blood volume by as much as
10 percent (Linderkamp et al., 1977). Consequently,
blood volumes determined using the Evans-blue dye
method may tend to be higher than those in studies
that use labeling techniques.
Evidence from the literature seems to confirm this
reported difference. For instance, the average blood
volume of rabbits in two studies using the Evans-blue
dye method is 70 ml/kg (Courtice, 1943) and 69.8 ml/kg
(Aikawa, 1950). Armin et al. (1952) used a labeling
technique and reported a blood volume of 57.3 ml/kg
for albino rabbits and 64.7 ml/kg for brown rabbits.
Breznock and Strack (1982) concluded that radiolabeling techniques cannot be used to interpret blood
volumes of nonsplenectomized cats with accuracy
because labeled red blood cells are sequestered within
the spleen. By measuring blood volumes in splenectomized cats, the investigators concluded that true
blood volumes are probably closer to 4 percent of
body weight rather than 6-7 percent as is commonly
reported in the literature.
Sato et al. (1985) found significant differences in the
bleeding volume (percentage of total blood volume)
resulting in similar mortality rates in rats of different

animal is rapidly bled to maintain extremely low blood


pressures. Bassin et al. (1971) argued that the Wiggers
dog model is inferior because it poorly resembles
clinical hemorrhage, and Loegering and Carr (1978)
reported that sustaining blood pressure at 40-45 mm
Hg by bleeding rats causes a more severe shock state
than reducing blood by a fixed volume. We mention
this model of hemorrhage in passing because of its
previous popularity, but we see little relevance in this
branch of blood loss research for the purposes of this
paper.

Various experiments have focused on different parameters to evaluate the effects of blood loss in humans
and animals. In the following section, discussions of
blood loss are organized according to these various
parameters. Table 4 summarizes results from the
literature on blood losses of greater than 30 percent
of total blood volume.
TABLE 4

Cardiac Output In studies on the effect of acute


blood loss on cardiac output in rats, a hemorrhage of
25 ml/kg caused an 85-percent reduction in cardiac
output (Saperstein et al., 1960). The 25 ml/kg hemorrhage, which by our calculations reduced total blood
volume by about 40 percent, caused mortality in all
animals in the study.
Arterial Blood Pressure Studies in dogs on the effects
of blood loss on arterial blood pressure suggest that if
blood pressure falls below a critical level, the animal
will die. In one study, dogs whose arterial pressure
fell below 45 mm Hg died; those whose arterial pressure
remained above 45 mm Hg survived (Guyton, 1963).

Effects on Animals and Humans of Blood Loss Greater than 30 Percent of Total Blood Volume

Species

Volume
Removed

% Total
Blood
Volume

Sample Site and Method

Results

References

85% reduction in cardiac


output

Saperstein et al., 1960

Rat

25 ml/kg

40

..."

Rat

6 ml over 5 h

50

Polyethylene cannula in
caudal artery

Unaltered biodegrad. of
quinidine sulf. if vol.
replaced immed. with
bood or saline

Agrelo and Miliozzi, 1974

Rat (germ-free)

2.6 ml/100 g
over 10 min

40 *

Jugular vein cannula

Critical bleeding volume

Yale and Torhorst, 1972

Rat (germ-free)

3.0 ml/100 g
over 60 min

47*

Jugular vein cannula

Critical bleeding volume

Yale and Torhorst, 1972

Rabbit

20-33 ml/kg
over 5 h

50*

Polyethylene cannula in
jugular vein

4- to 5-fold increase in
plasma levels of acid
hydrolases

Courtice et al., 1974

Dog

> 30

20 mm Hg or greater drop
in blood pressure, pulse
> 100

Tovey and Lennon, 1962

Dog

46

Arterial pressure fell to


29% of control; all dogs
died within 2.5 h

Furneaux, 1969

Transient decrease in
plasma prothrombin,
increased activity of
aminotransferases,
phosphatases

Glowinski et al., 1972

88% mortality

Hobler and Napodano,


1974

Compensatory
mechanisms are
inadequate

Simon and Olsen, 1969

Hemorrhagic shock

Noble and Gregerson, 1946;


Williams et al., 1983

Dog

566-766 ml at
50 ml/min
over 12-16
min

43.6
('/> body
weight)

Swine
(unanesthetized)

Bled over 30
min

40

Swine
(unanesthetized)

30 ml/kg,
rapid
withdrawal

40

Human

Femoral artery puncture

Superior vena cava


catheter

30-40

"Data not available.


''Estimated values based on Table 1 and/or experimental data.
Volume 31, Number 4

Fall 1989

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Absolute Volume Removal

Hemorrhagic Shock Studies that focus on clinical


signs of hemorrhagic shock agree that a blood loss of
30-40 percent of the total blood volume results in
hemorrhagic shock (Noble and Gregerson, 1946; Williams et al., 1983). Clinical signs of hemorrhagic shock
include pallor, cold skin and extremities, a fast thready
pulse, depression and restlessness, hyperventilation,
muscular weakness, and subnormal body temperature.

Residual Blood Volume A number of blood loss


studies in dogs have focused on residual blood volume,
that is, the volume of blood remaining one to two
hours after hemorrhage. This value takes into account
the body's ability to respond to blood volume loss by
mobilizing reserve blood from the spleen and fluid
from the interstitium. One study of dogs showed a 50percent survival rate associated with a residual blood
volume of 62.6 percent of the prehemorrhagic blood
volume and an 80-percent survival rate associated with
a residual blood volume of 70.3 ml per kg of body
weight, or about 72 percent of the prehemorrhagic
volume (Wang et al., 1947).
Walcott (1945) showed a residual blood volume of
60 percent to be the irreducible minimum compatible
with survival. Each dog in Walcott's study underwent
a single massive bleeding with a loss of about one-half
its total blood volume. In the group that survived,
residual blood volumes one hour after hemorrhage
averaged 65.5 percent of the prehemorrhagic volume,
and in the group that died, residual blood volumes
averaged 57 percent of the prehemorrhagic volume.
Thus, survival in this study correlated with the individual's ability to mobilize reserve plasma and cell
volumes.
Another study, done on splenectomized dogs, showed
a 50-percent survival rate to occur at a residual blood
volume of 66 percent (Rawson et al., 1959). All dogs
with residual volumes greater than 74 percent survived,
and all dogs with residual volumes of less than 62
percent died. Seven of 13 animals within the 62- to
74-percent range died. Incidentally, only in some
species does the spleen serve as a blood storage organ;
in others, such as mice and rats, the situation would
be more comparable to splenectomized dogs.
Mortality One study on germ-free rats focused on
critical bleeding volumes, that is, the critical blood
volume loss below which mortality increased sharply
(Yale and Torhorst, 1972). The critical bleeding volume
for blood withdrawn at a steady rate over 10 minutes
10

was 2.6 ml per 100 g or, by our calculations, roughly


40 percent of total blood volume. For blood withdrawn
over 60 minutes, the critical bleeding volume was 3.0
ml per 100 g, or roughly 47 percent of the total blood
volume. This study concluded that rate of blood loss
as well as amount of blood loss affects mortality.
Blood Values In dogs, a hemorrhage equal to 1/30 of
the dog's body weight, or according to the authors a
43.6-percent blood loss, caused increased activity of
aminotransferases and phosphatases, changes that are
due to hypotension and impaired tissue and organ
metabolism, and a transient decrease in blood prothrombin levels (Glowinski et al., 1972). Parenthetically, if we assume the total blood volume of a dog to
be 8.6 percent of body weight (Table 1), a loss of
blood equivalent to 1/30 of the body weight would be
38 percent of the total blood volume, a 5.6-percent
difference from the authors' calculation.
Moderate or Small Blood Losses

Table 5 summarizes results from the literature on


blood losses of 30 percent of total blood volume or
less. Like the last section, this one is organized
according to the various research parameters used to
evaluate blood loss. None of the studies described in
this section concern sustained hypotension; all deal
with absolute volume removal.
Cardiac Output

Saperstein et al. (1960) reported that a hemorrhage


of 10 ml/kg in rats caused a 50-percent reduction in
cardiac output. By our calculations, this loss is roughly
15 percent of the total blood volume.
Ploucha and Fink (1986) contrasted the effects of a
4-ml hemorrhage on 309-g rats and 233-g chickens.
This loss, about 20 percent of the rat's volume by our
calculations, reduced cardiac output in the rats by 43
percent, but reduced cardiac output in the smaller
chickens by only 4 percent. The authors explain the
chicken's superior ability to endure massive hemorrhage by its ability to mobilize extravascular fluids
twice as rapidly as the rat. This occurs despite the
fact that hemorrhage decreases, rather than increases,
peripheral resistance in the chicken.
Arterial Blood Pressure

In men who were bled 15 percent of their total blood


volume, arterial pressure dropped from an average of
92 mm Hg before hemorrhage to 76 mm Hg one minute
after hemorrhage. Arterial pressure then rose again to
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Unfortunately, the data published on these experiments do not include total blood loss and body weight,
so we cannot know what percentage of blood these
animals lost.
Furneaux (1969) observed that arterial pressure in
dogs who were bled 46 percent of their total volume
fell to 29.5 percent of the control just after the hemorrhage, rose to 45.3 percent of the control after two
hours, and then fell again as death approached. All
dogs in this study died.
According to one human study, when a drop in
blood pressure of 20 mm Hg or more corresponds with
a pulse greater than 100, blood loss is estimated to be
at least 30 percent of the total blood volume (Tovey
and Lennon, 1962).

82 mm Hg after 90 minutes (Skillman et al., 1971).


Ploucha and Fink (1986) reported that in rats, a 4ml hemorrhage, roughly 20 percent of the total blood
volume by our calculations, decreases arterial pressure
TABLE 5

by 25 percent. According to Furneaux (1969), dogs


that were bled 27 percent of their total blood volume
showed a return of arterial pressure to normal in less
than two hours.

Effects on Animals and Humans of Blood Loss Less than 30 Percent of Total Blood Volume
% Total
Blood
Volume

Rat

20 ml

30"

Rat

1-2 ml at 1
ml/7.5 min

5-11

Rat

3 ml at 1 ml/
7.5 min

Rat

Sample Site and Method

Results

References

Inferior vena cava


puncture

Severe gastric necrosis

Menguy et al., 1974

No change in plasma
corticosterone/prolactin

Wiersma and Kastelijn,


1985

16"

Increased plasma
corticosterone

Wiersma and Kastelijn,


1985

15"

50% reduction in cardiac


output

Saperstein et al., 1960

No change in plasma
prolactin (attributed to
slow sampling method)

Mattheij and van Pijkeren


1977

Rat

3 ml over 5 h

13"

Rat

1.2 ml over
20 min

6-7

Arterial catheter

Significant change in
plasma prolactin; no
change when volume
replaced by saline

Lawson and Gala, 1974

Rat
(unanesthetized)

4 ml

20"

Femoral artery catheter

Mean art. press, deer.


25%, cardiac output
deer. 43%, total
peripheral resistance
deer. 65%

Ploucha and Fink, 1986

Chicken
(unanesthetized)

4 ml

Ischiatic artery catheter

Mean art. press, deer.


15%, cardiac output
deer. 4%, peripheral
resistance deer. 13%

Ploucha and Fink, 1986

Dog
(unanesthetized)

26 ml/kg

Tygon catheter in jugular


vein

Sustained deer, in mean


art. press, of 23 mm Hg,
heart rate incr. by 89/
min, cardiac output fell

Vatner, 1974

30"

Dog

10

Arterial and venous


pressures returned to
normal within 2 h

Furneaux, 1969

Dog

27

Arterial and venous


pressures normal within
24 h, blood volume
normal after 90 min

Furneaux, 1969

17% mortality
66% mortality
66% mortality

Hobler and Napodano,


1974

Superior vena cava


catheter

No signif. change in
capillary pressure to any
tissue except stomach

Simon and Olsen, 1969

Tygon catheter in jugular


vein

Decrease in mean arterial


pressure of 23 mm Hg,
heart rate rose by
67/min

Vatner, 1974

"Venous removal"

Signif. deer, in cardiac


index, stroke vol., left
ventric. work, art.
press., central venous
press.

Skillman et al., 1971

Swine

Bled over 30
min; after
2 h, lost
volume
reinfused

10
20
30

Swine

15 ml/kg,
rapid
withdrawal

20

Baboon
(unanesthetized)

14 ml/kg

Human
(males)

800 ml
average
over 15.5
min

15

"Data not available.


''Estimated values based on Table 1 and/or experimental data.
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Species

Volume
Removed

Ulcers

In studies of hemorrhage in rats, Menguy et al.


(1974) reported that a hemorrhage equal to 2 percent
of the animal's body weight caused severe gastric
epithelial necrosis within 15 minutes and gross erosions
within 45 minutes. By our estimate, assuming the total
blood volume of a 300-g rat to be about 20 ml, a 2percent loss of body weight, or about 6 ml of blood,
is about 30 percent of the total blood volume.

Many studies of blood loss in humans focus on the


vasovagal fainting response (Barcroft et al., 1941; Ebert
et al., 1941; Grindon, 1982; Howarth and SharpeySchafer, 1947; Poles and Boycott, 1942), although the
literature does not address whether this response is
important to nonhuman animals or even occurs in
them.
Blood Values

Attempts to measure a distress response in animals


have been frustrated by the lack of a single predictable
measure of stress. The three systems activated in
response to stressbehavior, autonomic nervous system, and neuroendocrine systemall have been evaluated for use as measures of the stress response.
Unfortunately, indicators of an autonomic response,
such as plasma catecholamines, fluctuate too rapidly
and unpredictably to be of much help.
Behavioral responses are complex and vary from
one species to another, although studies of changes in
grooming and exploratory behaviors indicate that they
might be useful as distress indicators. Antin et al.
(1975) reported that grooming behavior is a normal
and predictable sequel of feeding. More recently,
Barclay et al. (1988) reported that they have developed
a reproducible and sensitive index of an animal's
reaction to various experimental procedures based on
exploratory behavior. They demonstrated that both
mice and rats explore a new cage and that the amount
of exploratory behavior is reasonably predictable in
the absence of disturbance. The state of the animal
following a particular procedure can then be assessed
by observing the departure (either excitation or depression) from the normal levels of exploratory behavior. The method is sufficiently sensitive to discriminate between the handling of mice by experienced or
inexperienced handlers. However, injections of nonirritant substances in modest amounts did not change
the behavioral response. Although the authors did not
assess the effects of various blood-sampling techniques
12

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Vasovagal Response

or regimens, the experiment would be an easy one to


do.
At present, a popular indicator of stress is fluctuation
in plasma concentrations of adrenal and hypophyseal
hormones. Unfortunately, not all stresses initiate a
change in corticosteroid levels (Breazile, 1987; Moberg, 1987), so these hormones may be overused as
indicators. However, a change does occur predictably
in response to certain stressors, such as injury, physical
restraint, and electric shock (Moberg, 1987). Krulich
et al. (1974) reported that of a variety of hormone
serum concentrations responsive to stress, including
prolactin, GH, luteinizing hormone, and follicle-stimulating hormone, prolactin concentrations are most
susceptible, because a significant response is consistently observed, even following very mild stimulation.
In one study, corticosterone and prolactin changes
appeared after roughly 15 or 16 percent of total blood
loss. The study concluded that rats bled 1 or 2 ml at
a rate of 1 ml per 7.5 minutes from an atrial cannula
(shown in the study to be a stress-free method of blood
sampling) were not stressed by the blood loss. However, those bled 3 or more ml at the same rate did
experience stress, because corticosterone levels increased significantly as one reached and exceeded the
3-ml level (Wiersma and Kastelijn, 1986). The study
offered no data on percentage of blood volume removed. The rats used in the study ranged in weight
from 180 to 430 g; therefore based on Table 1, we
estimate that a 3-ml blood loss would equate with a 9to 23-percent reduction of total blood volume, or a
mean value of 16 percent.
In another study, stress indicators appeared after as
little as a 6- to 7-percent blood loss. Lawson and Gala
(1974) reported significant responses of plasma prolactin concentrations after removal of only 1.2 ml of
blood from 225- to 300-g rats, or 6-7 percent of total
blood volume, within 20 minutes, and no changes in
prolactin concentrations when the lost blood was
replaced by saline. In the study, an indwelling arterial
catheter was used to eliminate stresses associated with
sampling technique.
Hematocrit has not proven to be a helpful indicator
of blood loss or physiological stress. Although a low
initial hematocrit predisposes an animal to early onset
of irreversible shock (Crowell et al., 1958), hematocrit
may not accurately reflect true red cell losses for up
to 72 hours because plasma and red cell volumes are
reduced proportionally during hemorrhage (Wintrobe
et al., 1981).
Some studies of humans donating a standard 400-ml
unit (about 8 percent of total blood volume) have
focused on length and magnitude of depletion of critical
factors. One study of iron deficiency concluded that,
after a single 400-ml (8 percent) loss, normal dietary
intake of iron may not return iron values to normal

until after 4 months in men and 8-12 months in women


(Finch, 1972). Another study reported that hemoglobin
concentration is lowest one to two weeks after donation
and reaches predonation levels three to four weeks
after donation (Wadsworth, 1955).
Physical Performance

Mortality

Surprisingly, in studies on anesthetized pigs, a 20to 30-percent blood loss is associated with significant
mortality. In one study, a 30-percent blood loss caused
66 percent mortality (Hobler and Napodano, 1974). In
the same study, a 20-percent loss also caused 66 percent
mortality, while a 10-percent loss caused 17 percent
mortality. These results should be interpreted in the
light of comparative studies of hemorrhage in anesthetized and unanesthetized pigs. One such study
demonstrated that a 20-percent blood loss in a pentobarb-anesthetized pig has biological effects similar
to a 40-percent blood loss in a nonanesthetized pig
(Simon and Olsen, 1969). In none of the studies on
animals other than pigs was mortality a factor with
volume losses of 30 percent or less.
Biotransformation

Dogs bled as little as 5 ml/kg, or about 6 percent of


their total volume, showed a measurable decrease in
the rate at which IV-administered hexobarbital was
biotransformed (Cumming et al., 1971). This effect is
due to decreased perfusion of the liver.

Recommendations for Single Blood Samples

From the literature on acute blood loss, one can


suggest that a 30- to 40-percent loss is too great: a 40Volume 31, Number 4

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A 1982 article by Grindon surveys studies on blood


donation in humans. One study cited by Grindon on
physical performance following blood donation in humans indicated a deterioration in performance lasting
a few days, particularly for activities requiring sustained endurance. According to a second study cited
by Grindon, maximum oxygen uptake levels, five days
after bleeding, were only 6 percent lower than predonation levels, which is probably due to the improved
oxygen unloading of hemoglobin. A third study concluded that performance and endurance levels completely return to normal only after 28 days.

percent blood loss is, at least for germ-free rats, the


critical bleeding volume (causing 50 percent mortality),
and a 30- to 40-percent loss corresponds with hemorrhagic shock. Because of a lack of data on stress
indicators below a 30-percent blood loss, we cannot
make absolute recommendations about smaller losses.
However, in one study, corticosterone levels may be
elevated in rats that undergo approximately a 16percent blood loss, while in another study, levels rise
after as little as a 6-percent blood loss. We tentatively
recommend that investigators limit blood sampling to
15 percent of total blood volume for a single sampling
and provide special justification for taking volumes
greater than 15 percent.
The Department of Laboratory Animal Medicine at
Cornell University recommends a 15- to 20-percent
limit with a 30-day recovery period. Given the absence
of reliable data in the literature, there is some danger
that the 20-percent limit may be too high because of
the potential errors described above in estimating blood
volume based on body weight.
How do our recommended limits compare with other
rules of thumb currently used by investigators? One
commonly used rule of thumb is referred to as the 10
percent-10 percent rule. This approach assumes that
a safe sampling volume is 10 percent of the total blood
volume, which is estimated to be 10 percent of the
animal's body weight. On the surface, the approach
seems more conservative than our own because it
recommends a 10-percent sampling limit. Actually, the
approach significantly overestimates the blood volume
of laboratory animals, so that calculated bleeding
volumes based on the 10 percent-10 percent rule are
equal to or greater than volumes calculated using blood
volume tables and our own 15-percent maximum sampling volume. For example, according to the 10 percent-10 percent rule, an investigator may take 2.5 ml
of blood in one sampling from a 250-g rat. Actually, a
rat's blood volume is closer to 6 percent of its body
weight (Table 1) than 10 percent. Applying our own
15 percent maximum recommended bleeding volume,
an investigator may take 2.4 ml from a 250-g rat, a
volume slightly less than the one reached with the 10
percent-10 percent rule.
Investigators might consider replacing lost blood
volume with saline to reduce the stress of blood volume
loss. Lawson and Gala (1974) reported that plasma
prolactin concentrations in ovariectomized rats decreased significantly after removal of only 1.2 ml of
blood within 20 minutes. However, when the lost
blood was replaced with saline, no change in prolactin
levels occurred. Also, Rochae Silvaet al. (1987) found
that severe hemorrhagic shock (44.5 ml/kg, or about
50 percent of total blood volume) was reversed by
intravenous administration of a small volume (4 ml/kg)
of NaCl solution.

BIOLOGICAL EFFECTS OF MULTIPLE


HEMORRHAGES

The data on multiple hemorrhages or samplings are


more sketchy, and thus recommendations for multiple
samplings are more difficult to offer.
Single Day

More Than One Day


Cardy and Warner (1979) reported that a monthly
sampling of 1.25-2.0 ml of blood from young mature
Fischer 344 rats caused no effect on common blood
values (hemoglobin, hematocrit, red blood cell count,
mean corpuscular volume [MCV], mean corpuscular
hemoglobin, mean corpuscular hemoglobin concentration [MCHC], total leukocyte count). However, the
protocol caused a decrease in the rate of body weight
gain. This difference was noted as early as 3 weeks
after the study began, was apparent in both sexes, and
persisted throughout the 23-week study. In the protocol, each rat was bled by orbital puncture less than
1 ml every two weeks. An average of 1.6 ml was taken
from each rat per month. This volume constitutes
roughly 25 percent of the total blood volume at the
beginning of the study, when all rats weighed about
100 g, and constitutes 7 percent of the total blood
volume of males (350 g) and 13 percent of the total
blood volume of females (200 g) at the study's end.
The authors concluded that "the regular withdrawal
of amounts of blood small enough not to induce
abnormal hematologic values can affect other parameters of physiologic status."
In one study of repeated blood withdrawal, six 4ml samples of blood were taken from rats at one- or
two-week intervals (Wiersma and Kastelijn, 1986).
Blood parameters in the first group (weekly blood
withdrawal) were significantly affected, while those in
the second were not. In the rats in the first group,
hemoglobin and MCHC values decreased steadily
while blood sedimentation values and osmotic resistance increased steadily. It should be noted that some
14

Recommendations for Multiple Samplings


We can offer very little in the way of conclusive
recommendations for multiple bleedings. The Wiersma
study made it clear that a loss of about 20 percent or
slightly more per week is too much, but studies of
acute bleeding discussed in the previous section suggest that these limits may be too great for even a single
sampling. The monthly sampling study by Cardy and
Warner (1979) resulted in significant growth depression, although it is difficult to identify this effect with
a specific percentage of blood volume removal because
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Agrelo and Miliozzi (1974) reported taking 6 ml of


blood, roughly 50 percent of the total blood volume
of a 175-g rat, over five to six hours in 1-ml samplings.
The authors reported no changes in biodegradation of
drugs if each 1-ml blood sample is replaced by blood
or saline. They also noted that their use of a caudal
artery cannula caused no tail necrosis. Upton (1975),
using a jugular canula, reported taking 15 blood samples
of 0.2 ml, or 3 ml total, over six hours from rats
without the rats showing ill effects.

of the indicators used in this study, such as MCHC


and hemoglobin, are relatively insensitive indicators
of stress. The researchers provided no data on percentage of blood volume lost, so we can only estimate
from the average weights of the rats (225 g for females
and 375 g for males) that between 17 and 28 percent
of the blood volume, or a median value of 23 percent,
was removed.
A study on rabbits by Nerenberg et al. (1978) focused
on the effects of chronic bleeding on several blood
components. For a period of eight weeks, rabbits in
the study were bled 1.5 percent of their body weight
three times a week, or about 50 percent of their total
blood volume per week. Parameters were measured
at the end of the 8-week period and again after a 45day rest period. At the end of the experimental period,
red blood cell, hemoglobin, and hematocrit levels were
low. After the rest period, all of these parameters were
higher than before the bleedings were begun, which
suggests overcompensation. Based on MCV and MCHC
values, all rabbits bled according to this protocol
developed macrocytic hypochromic anemia. Nevertheless, the authors recommended the protocol for the
production of large amounts of antisera.
A study of women who donated a standard unit
(about 400 ml, or 8 percent of total blood volume)
every two months without iron therapy found that,
after four donations, one-third of the women had no
stainable iron in the marrowthat is, their iron stores
had been depleted (Lieden, 1975).
One study that looked at hemoglobin as an indicator
of blood status concluded that improved oxygen unloading by hemoglobin in the weeks following bleeding
more than compensates for the reduced hemoglobin
concentration (Edwards and Cannon, 1972). This improved oxygen unloading seems to be due to the
increased number of young erythrocytes following
bleeding. However, it is uncertain from this study of
men who had donated 250 ml weekly for two weeks
or a single 500-ml unit, what effect multiple bleedings
beyond a period of a few weeks would have on oxygen
unloading and hemoglobin concentrations.

SAMPLING TECHNIQUES

A 1983 article, with over 500 references, reviews


the literature from 1952 to 1982 on techniques for
vascular access in the rat (Cocchetto and Bjornsson,
1983). The article describes 39 procedures for arterial
samplings from 7 sites, 81 procedures for venous
sampling from 19 sites, and 4 "miscellaneous" procedures that yield a mixture of arterial and venous
blood. The article includes expected blood yields from
some of the more common procedures. For instance,
retroorbital plexus collection is described as yielding
about 0.5 ml of venous blood, tail amputation as
yielding up to 4 ml of mixed venous-arterial blood,
and cardiac puncture as yielding up to 5 ml of mixed
blood.
Our intention in writing this section was not to sift
through the vast body of literature on sampling techniques for the purpose of offering absolute recommendations on which technique to use on each animal.
Researchers have at least as many reasons for collecting blood as there are sampling techniques, and
each technique is more suited to some purposes than
others. Furthermore, in the judgment of some researchers whose opinions we solicited, sampling techniques considered stressful by some (e.g., retroorbital
puncture) in fact cause minimal stress in the hands of
technically adept handlers.
The brief survey of sampling techniques below aims
to report facts and opinions regarding sampling techniques, particularly those concerned with stresses to
animals. We hope it will serve as a helpful resource.
Rats

In the rat, many techniques are used for sampling


blood. The three most common are retroorbital plexus
Volume 31, Number 4

Fall 1989

puncture, tail amputation, and heart puncture.


Retroorbital plexus puncture yields an average of
0.5 ml of venous blood in the rat (Cocchetto and
Bjornsson, 1983). Grice (1964) described a procedure
for bleeding the medial canthus that yields 1-2 ml of
blood. When the technique was first described, yields
of up to 1 ml were sampled from the anterior (medial)
canthus (Stone, 1954). According to Sorg and Buckner
(1964), sampling from the posterior (lateral) canthus
causes fewer nose bleeds and less trauma to the eye
and is useful for repeated samplings in rats, mice,
guinea pigs, rabbits, and hamsters. However, these
same authors reported success in taking up to 8 ml of
blood from a rat with retroorbital plexus puncture
without killing the rat. Even in a 400-g rat, 8 ml of
bloodabout 36 percent of the total blood volume
exceeds limits we would recommend for a single
nonterminal bleeding.
Descriptions of the retroorbital puncture method
commonly include the following steps: making the eye
protrude by retracting the skin adjacent to the eye,
causing constriction of venous return by applying
thumb pressure behind the angle of the jaw, and
inserting a pipette gently through the canthus into the
retroorbital plexus, gently rotating the pipette as it is
advanced (Kraus, 1980). However, it has been reported
that these steps are sometimes not practiced, and
instead the pipette is more or less inserted behind the
orbit and scraped through the orbital plexus, a procedure that probably causes serious hemorrhage but
is likely to yield larger blood volumes.
An article by Timm (1979) challenges the conventional assumption that the rat has an orbital plexus.
Instead, Timm's study suggests that rats have a venous
plexus. The paper has several implications for orbital
bleeding:
The medial canthus is not a proper site for orbital
bleeding because it is occupied largely by the Harderian
gland and has only small anastomotic veins.
The area ventral to the orbit is also not an
appropriate site since it has a variable anastomotic
vein.
The ophthalmic venous plexus is difficult to reach
with a capillary tube because it lies deep in the orbit.
The area dorsal to the orbit is the most appropriate
site for sampling because it is occupied by the caudaldorsal anastomotic vein. The article describes a procedure for sampling from this site.
Using cardiac puncture, Burhoe was able to collect
5 ml of blood from rats weekly for three months
(Burhoe, 1940). Another study reported 12 percent
fatality for heart puncture (Stuhlman et al., 1972).
Incidentally, it is becoming a standard requirement of
many institutional animal care and use committees
that cardiac puncture be used under anesthesia and
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the animals were gaining weight throughout the study.


During the initial stages, approximately 25 percent of
the blood volume was being removed. Cornell University recommends a 10-percent weekly limit, but
perhaps a 7.5-percent weekly limit would be more
prudent given the evidence that hemoglobin concentrations may take many weeks to return to normal
following a loss as small as 8 percent. In written
correspondence, W. J. Dodds (New York State Department of Health, personal communication, 1988)
concurred with a 7.5-percent weekly limit. Dodds'
own research on platelet and coagulation requires
measurement of very sensitive blood parameters, so
that stress and physiological variables must be controlled. Her own bleeding limits are 10 percent of total
blood volume followed by a rest of at least two weeks.

Mice
In the mouse, retroorbital sinus puncture and heart
puncture are the most common sampling techniques,
although success has been reported with other methods, including severing brachial vessels, jugular vein,
carotids, femoral vessels, and abdominal aorta, as well
as venipuncture of the caudal vena cava and tail
vessels.
Cardiac puncture in the mouse yields variable blood
volumes, and the blood is likely to be hemolyzed and
contaminated by tissue fluids (Adeghe and Cohen,
1986). Anesthesia is required for cardiac puncture
(Moreland, 1965).
Retroorbital sinus puncture can yield up to 1 ml of
venous blood in the mouse (Moreland, 1965). Adeghe
and Cohen (1986) reported that many trials are required
16

for acquisition of an appropriate level of skill, that the


blood is likely to be hemolyzed, and that "the procedure is distasteful to many workers."
Cate (1969) described an orbital sinus sampling
technique for unanesthetized mice that yields about
50 percent of total blood volume. The procedure was
developed as an alternative to standard orbital sinus
sampling techniques, which typically yield small blood
volumes. The described technique, which involves
inserting polyethylene tubing in the medial canthus,
yields about 3 percent of total body weight, or 50
percent of total blood volume, in less than one minute.
Two recent letters to the ACLAM Newsletter argued
that orbital bleeding in the mouse is obviously painful,
and therefore, the technique should be accompanied
by anesthesia (Letscher, 1987) or avoided altogether
(Farnell, 1987).
Rabbits

In rabbits, the most common sites for blood sampling


are the marginal ear vein, the central artery of the ear,
and the heart.
Marginal ear venipuncture frequently is followed by
vascular spasm (Fick and Schalm, 1986; Tillman and
Norman, 1983). Incision of the marginal ear vein or
the central artery of the ear can yield up to 50 ml of
blood (Moreland, 1965). According to Moreland, anesthesia is not required for either procedure. It has been
fairly common to swab the marginal ear vein with
xylene to facilitate blood flow and collection, but
xylene can cause serious inflammation if not carefully
washed off. The policy set by the Department of
Laboratory Animal Medicine at Tufts/New England
Medical Center is to give the rabbit a dose of acepromazine IV in the marginal ear vein and collect the
blood from the central ear artery (M. Ellenberger,
personal communication, 1989). Grice (1964) reported
that 25 ml of blood, about 14 percent of total blood
volume, can be readily obtained from one auricular
artery.
Hoppe et al. (1969) described a technique for bleeding the marginal ear vein that can yield from 30 to 50
ml of blood (roughly 17-28 percent of total blood
volume) in four or five minutes. The technique, which
uses a vacuum reservoir and centrifuge tube to increase
sampling volume, is recommended for repeated samplings. In the author's words, "the technique of bleeding rabbits described here does not endanger the life
of the donor provided the animal is not excessively
exsanguinated."
Lumsden et al. (1974) described a modified technique
for orbital sinus sampling in the rabbit. Rather than
using as a sampling site the medial or lateral canthus,
which failed to yield consistent samples, the authors
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only as a terminal sampling procedure in all laboratory


species.
Bober (1988) noted that tail amputation has "fallen
from favor due to the traumatic nature of the procedure." Bober described a technique for sampling from
3 to 6 ml of arterial tail blood under general anesthesia
using a 22-gauge syringe.
Golba et al. (1974) concluded that sampling blood
from the retroorbital plexus of the rat is much more
stressful than sampling blood by tail amputation. Retroorbital sampling, but not tail amputation, caused a
significant decrease in white blood cell count for several
weeks. According to the authors, the decreased white
blood cell count indicates a general adaptation stress
response. This response to internal and external factors
results in lymph system atrophy and decreased mitotic
activity in bone marrow.
In another study comparing stresses of different
sampling techniques, Horton et al. (1986) observed
serum creatine kinase to be significantly higher following retroorbital plexus puncture but not following heart
puncture. The increase in creatine kinase levels could
be due to either tissue destruction or stress.
According to the studies above, retroorbital plexus
puncture seems to be the most stressful of the three
techniques discussed, although the high rate of fatality
reported for heart puncture does not recommend that
technique for nonterminal procedure either. Also,
repeated samplings from the retroorbital plexus should
be avoided because they cause local tissue damage
involving the Harderian gland (Canadian Council on
Animal Care, 1984).
Toe clipping under anesthesia has been used to
obtain small yields of blood, up to 0.3 ml, in the rat.
However, due to pain in the rat following the procedure, Cocchetto and Bjornsson (1983) did not recommend toe clipping.

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described a technique involving capillary tube insertion


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Saperstein, L. A., E. H. Saperstein, and A. Bredemeyer. 1960.


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Commentary: H. Richard Adams


The article by McGuill and Rowan provides a good
overview of what seems to represent, at first glance,
a rather straightforward and biologically unambiguous
topic about blood sampling in laboratory animals.
However, as is quickly emphasized, the issue of
sampling for hematologic assay in biomedical research
is far from simplistic and demands greater consideration than just the dexterity of venipuncture technicians.
One aspect of particular importance is the underlying
theme that blood-sampling techniques can evoke rather
substantial biologic reactions in the experimental animal. Such reactions alter the basal homeostasis of the
subject and thereby comprise yet another variable in
a research protocol. The sympathetic division of the
autonomic nervous system is especially reactive to

H. Richard Adams is chairman of the Department of Veterinary


Biomedical Sciences, College of Veterinary Medicine, and professor
in the Department of Pharmacology, School of Medicine, University
of Missouri-Columbia.
Volume 31, Number 4

Fall 1989

stressful environmental stimuli, and its activation during blood sampling can rapidly modify cardiovascular
dynamics and also influence metabolic functions of
other tissues and organ systems. Blood and its various
components themselves can be altered by blood collection procedures and associated paraphernalia,
whether sampling is acute or chronic. Some of the
resulting influences may well alter the precise hematologic variable undergoing study, some represent
general systemic stress factors that seem mostly unavoidable owing to the fact that blood sampling is
inherently invasive and traumatic, some are simply
not known or recognized, and some can be catastrophic
to the experimental study. Indeed, McGuill and Rowan
poignantly point out that one attempt to develop an
arterial catheter for chronic blood sampling in rats
actually yielded "an excellent model for arterial embolism" with intestinal and renal infarctions! The
importance of such confounding influences to certain
studies, especially if they remain unrecognized and
uncontrolled, can hardly be overemphasized.
Many blood-sampling factors now are known to hold
real potential for altering hematologic and other phys19

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Courtesy Orin B. Mock, Kirksville College of Osteopathic Medicine, Kirksville, Missouri.

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subsidiary elements of the entirety of this article, but


they illustrate two important caveats:
1. Judiciousness should be employed when single
literature sources are used to build specific policy
dealing with complex biologic problems.
2. Hemorrhage and blood-sampling procedures do
not constitute a trivial biologic problem.
The influence of chemical restraint during hematologic sampling is introduced relatively early in the
paper. Yet in later discussions of hematologic variables, the reader is not informed if the data originated
from anesthetized, sedated, or conscious animals.
Acclimation to handling and environmental conditions
are addressed as important issues in all species, and
yet, later comments about pertinent data in the literature do not distinguish between trained versus naive
subjects. These limitations should not be criticized as
weaknesses unique to this review. Rather, they serve
to substantiate the dearth of information available from
some of the original reports and the resulting lack of
historical background on precise biologic consequences of blood loss and sampling. In relevant literature about hematologic sampling in the past, details
that are now deemed important were not always
recognized as salient features at that time. It seems
quite likely that currently undiscovered sequelae of
blood loss will turn out to be important considerations
in the future.
In summary, the article by McGuill and Rowan
contains important generic and starting-point information about blood-sampling techniques, as well as
relevant guidelines about total blood volumes versus
volumes withdrawn for hematologic samples in different species. This information should guide investigators about this issue, but it does not absolve them
from the responsibility to validate and justify the
appropriateness of their own hematologic assay procedures relative to their own particular experimental
objectives.

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iologic variables. These factors include methods of


physical restraint, types of chemical restraint drugs,
blood volume removed, subject experience, and investigator experience. These and other related and
unrelated influences are addressed by McGuill and
Rowan. A strength of their review is that it tabulates
a selected sampling of a broad topic entailing even
more complexity than could be assembled in this
setting. Another value of this article is that it incorporates related animal care and welfare issues the
biomedical community must consider. Thus, on the
one hand, this article is an appropriate starting point
for investigators and laboratory animal veterinarians
needing a literature resource for information about
blood-sampling technology and its affiliated intricacies.
On the other hand, this review should not be misconstrued as a definitive treatise or policy directive
about precise blood-sampling techniques and resulting
biologic reactions. The authors make no such claim,
and it would be a mistake for readers to rely on such
an assumption. This topic is too complex to justify
such a universal application, as revealed by the paper
itself. The sections dealing with hemorrhagic shock,
for instance, are superficial relative to the data base
in this field, and they actually are somewhat tangential
to the main points of the article. Readers could be
mislead by the comment that severe hemorrhagic shock
evoked by removal of 50 percent of total blood volume
in dogs is reversible by a small volume (4 ml/kg) of
"NaCl solution.11 The study in question actually used
a highly hyperosmolar solution of NaCl, a major
distinction from isotonic NaCl solution. Responses of
the sympathetic nervous system to hemorrhage or
other stressors are mediated not by "cholinergics,11
but by the adrenergic mediators norepinephrine and
epinephrine. Review/advisory committees or consultants could find themselves in a rather untenable
position if they relied solely on this article for detailed
information about saline resuscitation or nervous system responses to hemorrhage. These points reflect

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