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1. The Effect of Anemia on Wound Healing
CHRISTOPHER HEUGHAN, B.A., M.B., B. CHIR., F.R.C.S.,*
GUNTA GRISLIS, B.A., THOMAS K. HUNT, M.D., F.A.C.S.
ANEMIA FREQUENTLY IS BLAMED for defective wound
healing.3,7,11,25 Most young surgeons today are
taught that normal healing is jeopardized when the hema-
tocrit falls below 35%. Each year countless blood transfu-
sions are given to meet this arbitrary and questionable
criterion despite the fact that it is supported neither by
clinical nor experimental fact.
Three of 4 clinical surveys of patients with abdominal
wound disruption have failed to implicate anemia as a
major factor. Marsh,19 Mann,'8 Alexander2 and their
co-workers found that anemia, uncomplicated by other
deficiencies, did not contribute significantly to wound
dehiscence. Guiney and his colleagues,8 by contrast,
found that 50% of their patients who disrupted their
abdominal wounds were anemic as opposed to 20% of
the control group who healed normally. However, their
criterion of anemia, a hemoglobin of less than 12 gm/100
ml., was somewhat strict.
The results of experimental studies, without close
analysis, appear conflicting. Besser and Ehrenhaftb found
no decrease in the bursting strength of stomach wounds in
dogs made anemic by bleeding and retransfusing pooled
plasma. Trueblood and his co-workers23 found no de-
crease in the bursting strength of colonic anastomoses in
rats made anemic by iron deficiency or bleeding and
retransfusion of plasma. Adamson' showed no difference
in skin bursting strength with bleeding and retransfusion
with plasma expander.
Results obtained by other investigators prove that
nutritional deficiency incidental to the production of
Submitted for publication March 5, 1973.
Supported in part by USPHS No. GM12829 and USPHS No.
GM18470.
* Present address: Department of Surgery, Memorial University
of Newfoundland, St. John's, Newfoundland. Canada.
Reprint requests: Thomas K. Hunt, M.D., Dept. of Surgery,
University of California Medical Center, San Francisco, Calif.
94143.
From the Department of Surgery, University of California
School of Medicine, San Francisco, California and Trauma
Center, San Francisco General Hospital,
San Francisco, California
anemia accounts for some of the apparently conflicting
results. Waterman, et al.24 confirmed that anemia had
no effect on either the contraction of open wounds or
on the bursting strength of laparotomy incisions in young
rats. They also showed that control as well as anemic
rats fed powdered milk (a diet often used to produce
iron-deficiency anemia) with and without added iron,
showed slower contraction and reduced breaking strength
when compared to animals fed normal chow. The chow-
fed animals gained twice as much weight in the course
of the experiment. Macon and Poriesl7 also found that
iron deficiency anemia had no effect on wound breaking
strength and young rats fed on regular chow gained
both body weight and wound tensile strength more
rapidly than animals which were fed powdered milk
with or without iron. Nutritional depletion, therefore,
can explain the findings of Jacobso-n and Van Prohaskal5
whose chow-fed control mice showed a significantly
higher breaking strength than the anemic group which
was fed on an iron-free powdered milk diet. Bains,
Crawford and Ketcham4 reported decreased breaking
strength in young rats (145-165 gm) made anemic by a
combination of iron deficient diet and bleeding. How-
ever, iron deficiency in the young has a potent effect on
growth rate and hence on repair.
Hugo and his colleagues12 found a decrease in break-
ing strength in rabbits made anemic by hemolysis
with intraperitoneal phenylhydrazine after 6 days, but
there was no difference in strength at 9 or 12 days.
The toxic potential of phenylhydrazine on wound me-
tabolism is not known.
Abnormalties of blood volume or viscosity, both of
which severely impair healing, may also co-exist with
163

2. HEUGHAN AND OTHERS
anemia. Sandblom21 found decreased breaking strength
in wounds of rabbits made anemic by bleeding. These
animals were often dehydrated and hypovolemic. He
later showed that dehydration can decrease wound
strength. Sandberg and Zederfeldt22 repeated these ex-
periments but replaced blood volume with dextran and
restored healing towards normal. When they denervated
the wounded area, healing became entirely normal. Hunt,
et al. demonstrated that hypovolemia severely decreases
oxygen supply to the wound,14 and wound hypoxia de-
lays repair.6,13
Most of these studies relied upon measurement of
breaking strength as the index of repair. Collagen ac-
counts for the strength of wounds, and therefore any
factor which affects the strength of early wounds must
do so by interfering with collagen biosynthesis, or by
accelerating collagen lysis. Collagen synthesis requires
oxygen for several of its steps. Assembly of the amino
acid chains on the ribosome is energy-dependent. Fibro-
blasts contain the enzymes for glycolysis, but most of
their energy is ultimately derived from oxidative me-
tabolism. Hydroxylation of some of the proline and lysine
molecules, which is essential for structure and function
of the collagen molecule, requires vitamin C, ferrous iron,
a-ketoglutarate and molecular oxygen as essential co-
factors for the hydroxylating enzyme.'6
Under normal conditions the rate-limiting factor in
collagen biosynthesis is the local availability of molecular
oxygen. Niinikoski20 and Hunt and PaiI3 have shown
that the rate of gain in strength and collagen content in
experimental wounds was greater when the animals
breathed 45% to 60% oxygen and was less in an atmosphere
of 12% oxygen as compared to control animals breathing
air.
Obviously, oxygen is vital to healing, but the accumu-
lated evidence heavily favors the conclusion that uncom-
plicated or moderated anemia does not affect wound
healing. Nevertheless, surgeons continue to believe and
teach that it does. Presumably the reason for this is the
fundamental belief that red cell deficiencies must in-
evitably decrease oxygen delivery to the wound. Ap-
parently this belief will persist until it is shown conclu-
sively that anemnia does not decrease wound oxygen
supply. The present study was undertaken, therefore, to
demonstrate directly whether a mild, normovolemic
anemia impairs oxygen delivery to experimental granula-
tion tissue in the rabbit and secondarily, in a particularly
discriminating model, whether this degree of anemia
impairs accumulation of connective tissue.
Materials and Methods
Four stainless steel wire mesh cylinders were implanted
subcutaneously in each of 25 New Zealand White male
rabbits weighing 2 to 3 kg. After the cylinders had been
implanted, approximately 12 ml. of blood were removed
from every animal by cardiac puncture while anesthesia
was continued. The red cells were separated by centrifu-
gation and the serum was stored in a freezer.
Cardiac blood was withdrawn with a heparinized plas-
tic syringe from a 20-gauge 132 inch needle introduced
into the xiphosternocostal angle about 30 from the hori-
zontal and near the sagittal plane. This procedure can
be done with the animal under mild sedation.
On day 11, wound fluid was aspirated from the wound
cylinder and its oxygen tension and pH were measured.*
The pCO2 was determined by the Astrup method. This
method of wounding, fluid sampling, and measurement
has been described in detail by Hunt et al.'3
The animals were divided into anemic and control
groups, both of which were bled again by cardiac punc-
ture 12, 13, 18 and 20 days later. Twelve to 20 ml. of
blood were removed on the first 2 occasions and 24 to
30 ml. on the last. The serum from the anemic group
was separated by centrifuging. Pooled plasma was made
up to a volume equal to that of blood removed and was
reinjected into an ear vein. The control animals were
bled and retransfused with their own blood after a
similar time lapse to that required for separating the
plasma in the anemic group.
The pooled plasma was tested for agglutination of
the recipient red cells of 6 animals at the begnning of
the experiment. In no case was the reaction different
from a control slide of red cells and serum from the same
animal.
Blood for hematocrit and viscosity determinations was
taken from an ear artery. Viscosities were measured with
a cone-plate microviscometert at 4 different shear rates.
These determinations required only a total of 2 ml. of
blood, and did not add significantly to the anemia.
The animals were killed with an overdose of pento-
barbital on the twenty-second day and the wound cyl-
inders were removed and opened. The connective tissue
within was scraped off, defatted in three changes of 2/1
diethyl ether/acetone mixture and dried in an air oven
to a constant weight.
Results
Several rabbits died from hemothorax after cardiac
puncture. Several other animals in the group from which
red cells were withdrawn failed to show a change in
hematocrit, presumably because of concurrent dehydra-
tion. The data from these animals were discarded. Nine
anemic animals and 8 controls formed the basis of the
final data.
The packed cell volumes (P.C.V.) and wound fluid
* Radiometer gas monitor, Copenhagen N.V., Denmark.
+ Brookield Engineering Laboratories Inc. Stoughton, Mass.
Model LVT.
164 Ann. Surg. -
February 1974

3. THE EFFECT OF ANEMIA ON WOUND HEALING 165
oxygen tensions are summarized in Fig. 1. The mean
P.C.V. in the control group remained steady at 38%. The
P.C.V. of the anemic animals in the last period of the
experiment, when collagen is synthesized most rapidly
in this model, was 30%. The differences in P.C.V. be.
tween the anemic and control groups were statistically
significant on days 15, 18, and 22 (Fig. 1 ).
Wound oxygen tensions were identical in both groups.
The possibility that the oxygen tensions of the 2 groups
could significantly differ on any day is less than 2%. Car-
bon dioxide tension and pH were also identical (Table 1).
Dry weights of new connective tissue are also shown
in Table 2. The anemic group produced 12% more con-
nective tissue by dry weight than the control. This dif-
ference is significant (p < 0.025). In previous work we
have shown that dry weight of this tissue is an excellent
measure of collagen content. The hydroxproline content
of this tissue is constantly 44 !ug/mg collagen. Several
"spot samples" of tissue in this experiment showed this
ratio in both groups.
The mean whole blood viscosity at a shear rate of 1.15
seconds-' in controls was 6.4 + 0.4 (S.E.) centapoise and
remained stable throughout the experiment. As soon as
the anemia was induced, viscosity fell 19% to 5.2 + 0.3
centapoise. This was a significant difference, p < 0.02.
The differences at the 3 higher shear rates were also sta-
tistically significant.
Discussion
The role of dead space gas tensions in reflecting the
oxygen economy of an experimental wound has been de-
scribed by Hunt and his associates.13 Oxygen diffuses
from the capillary loops and is extracted by the wound
cells, therebyimaintaining a large gradient across the
wound edge. The oxygen tension of the wound fluid in
the "dead space" represents the lowest average extra-
cellular p°2 in the wound.13 The magnitude of this gradi-
ent is dependent upon the mean capillary pO2 (and
therefore upon the arterial oxygen tension) as well as
upon the rate of oxygen extraction by the wound cell
population for respiration, protein synthesis and proline
hydroxylation.
The mean capillary pO2 is maintained by transfer of
oxygen from red cells to plasma. This process in turn
depends on the oxygen content of the red cell, on the
efficiency of the transfer of oxygen from red cell to
plasma, and on the rate of flow of red cells through the
wound capillaries.
In this study the red cell density of the general circu-
lation was reduced in the anemic group. However, the
decrease in whole blood viscosity and consequent in-
crease in cardiac output tend to increase the rate of
passage of red cells through the wound. Furthermore,
the transfer of oxygen from the erythrocytes to the plasma
is likely to become more efficient due to an increase in
intracellular 2:3 diphosphoglycerate (2:3 D.P.G.) con-
centration. These are normal physiological responses to
anemia and tend to restore normal oxygen delivery. These
factors are apparently partly responsible for the increase
in connective tissue synthesis in anemic animals.
If anemia were to diminish the oxygen supply to the
extracellular fluid adjacent to the wound capillaries, one
of 2 things would have to happen: 1) the quantity of
oxygen extracted by the cells would remain constant,
in which case cell function would be maintained and
healing would proceed normally. Less oxygen would
then remain to diffuse into the dead space and the pO2
there would drop. 2) Alternatively the oxygen consump-
TABLE 1. Anemia
Day
11 15 18 22
anemia 39 1 2 39 i 1 38 41:
Hct. 39 4 1.7 * -7-_* -_*
control 32 :+ 1 34 4 1 30 i 1
anemia 91 2 10 1 2 10 4 2
PO2 8.3
control 10 42 11 d2 9 42
anemia 7.09 7.09 7.12
pH 7.09
control 7.11 7.08 7.11
anemia 72 68 66
pCO2 66.3
control 67 68 67
anemia 240 ± 10
Dryweight mg. mg*
control 213 4: 10
* Statistically significant (p < 0.025).
All other differences are not significant.
Vol. 179 * No. 2

4. HEUGHAN AND OTHERS166
0---0-O = ANEMlA
_^I-W = CONTROL
BLEEDING
; Volume Replacement
12 r
mmHg 10
P02
81-
45 r
FIG. 1. The changes in
packed cell volume and
wound-fluid oxygen ten-
sion are shown. The
oxygen tensions in the
two groups are statistic-
ally not different, but the
differences in packed cell
volume are significant on
days 15, 18, and 22.40 H-
Hct
35H
30'
11 15 18
DAYS AFTER WOUNDING
tion of the wound cells would decrease with the supply,
causing the oxygen gradient across the wound to become
less steep. In this case the oxygen tension of the wound
fluid might remain constant, but the weight of tissue
synthesized in the chamber would inevitably diminish.6
Experimentally, however, the pO2 of the dead space re-
mained constant, and the tissue synthesis was increased
by the anemia. Therefore, neither of these possibilities
occurred. Obviously, anemia did not diminish oxygen
supply. Arterial pO2, not oxygen content, is the quantity
which overwhelmingly governs oxygen supply to the
wound. This is not a revolutionary concept. It merely
recognizes that vascular damage has occurred in the
wound and that oxygen supply is, therefore, limited by
the rate at which oxygen can diffuse across the avascular
space of the wound. Pressure, not volume of gas, de-
termines the rate of diffusion.
The experimental model used in this study has pre-
viously demonstrated the effects of an impaired oxygen
supply under several experimental circumstances. Di-
minishing arterial PO2 from normal to 45 mm Hg lowers
dead space pO2 from 10 to 7 mm Hg and significantly
lowers connective tissue accumulation. Raising blood
viscosity by Dextran 500 infusion also produces a sig-
nificant fall in p02 and tissue synthesis.'0 Obviously this
model is capable of detecting subtle changes in oxygen
supply.
The present study demonstrates that mild, uncompli-
cated, normovolemic anemia, which commonly occurs
in surgical patients undergoing an otherwise uneventful
recovery does not require blood transfusion, with its at-
tendant dangers, to achieve optimal healing. This prob-
ably does not apply to severe anemia in which other
deficiencies may co-exist, or in which the compensatory
mechanisms may be overwhelmed or impaired.
Common sense dictates that at some point, anemia
inevitably will impede wound healing. This point has
not been determined precisely and may be in the range
of a P.C.V. of 15 according to Zederfeldt.26 Recently,
Adamsons' has shown that a hemoglobin of 5 to 6 gms.
(P.C.V. approximately 20) fails to impede repair in ani-
mals.
Summary
The existing evidence linking "anemia" with healing
is critically reviewed. Experimentally, no significant
change occurred in the oxygen economy of experimental
wounds in normal rabbits made anemic by bleeding and
retransfusing plasma. Connective tissue synthesis was
slightly greater at a P.C.V. of 30% than at a P.C.V. of 40%.
22
oo...
Ann. Surg. -
February 1974
I I I

5. Vol. 179 * No. 2 THE EFFECT OF ANEMIA ON WOUND HEALING 167
Healing is impaired by conditions which are often as-
sociated with anemia such as malnutrition, abnormalities
of circulating blood volume, and increased blood vis-
cosity following trauma. However, mild or moderate, un-
complicated, normovolemic anemia in otherwise healthy
individuals does not impair delivery of oxygen to the
wound and is of no consequence to wound healing. An-
ticipated stimulation of wound healing is not reason for
transfusion to correct mild or moderate normovolemic
anemia.
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