Oxygen Saturation Measurements in Canine Blood
Containing Hemoglobin Glutamer-200 (Bovine):
In Vitro Validation of the NOVA CO-Oximeter
Jonathan S. Jahr, MD; Bernd Driessen, DVM, PhD; Fedor Lurie, MD, PhD; Zuping Tang, MD; Richard F. Louie; Gerald Kost, MD
Abstract: This study was designed to validate in vitro oxygen saturation (SO2) measurements with the NOVA
CO-Oximeter (Nova Biomedical Corp, Waltham, Mass, USA) in canine blood containing hemoglobin (Hb) glutamer-200 bovine (Hb-200; Oxyglobin, Biopure, Cambridge, Mass, USA) as a Hb-based oxygen carrier recently
introduced into clinical practice. In the first set of experiments, stored blood from 6 mixed-breed canine blood
donors was used. Target PO2 levels were reached in aliquots of blood samples by tonometry. Oxygen saturation
was then measured with the test device and calculated based on known PO2 values. In the second set of experiments, total oxygen content was directly measured by means of an oxygen-specific electrode in aliquots of
fresh whole arterial, venous, and mixed (arterial-venous) blood withdrawn from the same canine blood donors.
Hb-200 was added to those blood samples to yield plasma Hb concentrations of 1.62, 3.25, 6.50, and 9.75 g/dL.
Based on Hb content and SO2 measured by the NOVA CO-Oximeter in these samples, total oxygen content was
also calculated for each sample and compared with measured values. A strong correlation was found between
SO2 values measured with the co-oximeter in samples after tonometry, and calculated SO2 based on known PO2.
Directly measured total blood O2 content varied by ≤ 5% from values computed based on co-oximeter measurements of Hb content and SO2. These results did not change with different levels of oxygenation of the samples or different plasma Hb-200 concentrations. In conclusion, the NOVA CO-Oximeter is an accurate analyzer
for measurement of SO2 after Hb-200 administration to canine blood. (Vet Clin Pathol. 2001;30:39-45)
©2001 American Society for Veterinary Clinical Pathology
Key Words: Hemoglobin-based oxygen carrier, hemoglobin glutamer-200 (bovine), NOVA CO-Oximeter, oxygen content, oxygen saturation, tonometry
———◆———
In both human and veterinary medicine, transfusion of
allogeneic blood has long been the mainstay in the
treatment of acute blood loss and anemia despite serious concerns associated with its use, such as transmittable diseases, immunologic incompatibility, transportation and storage difficulties, short shelf life, and
supply shortage.1-3 Recently, allogeneic and xenogeneic,
stroma-free, ultrapurified hemoglobin (Hb)-based oxygen carriers (HBOCs) have been developed as blood
substitutes to overcome these problems.4-6
Hemoglobin glutamer-200 bovine (Hb-200; Oxyglobin, Biopure, Cambridge, Mass, USA) is an ultrapure
solution of highly polymerized bovine Hb7,8 that has
been approved by the Food and Drug Administration
for treatment of anemia in dogs.9 It shares almost identical properties with Hemoglobin-Based Oxygen
Carrier 201 (HBOC-201; Hemopure), produced by the
same manufacturer as a proposed human oxygen carrier.7-9 Laboratory studies in various animal species
including the dog have demonstrated the efficacy of
these blood substitutes as volume expanders and oxygen carriers (for review see references).7,8,10-13 Moreover,
recent reports regarding the use of Hb-200 in veterinary
practice seem to confirm its efficacy in dogs.14-17 The
increasing use of Hb-200 and possibly other soon-available HBOCs in veterinary and human clinical practice
makes it important to validate the proper function of
oximetry devices after administration of blood substitutes.
Hemoglobin oxygen saturation (SO2) is usually determined by co-oximetry or blood gas analysis or both.
Blood gas analyzers measure pH, oxygen tension (PO2),
and carbon dioxide tension (PCO2). Hemoglobin saturation is then calculated with these values based on a stan-
From the University of California–Davis, School of Medicine, Departments of Anesthesiology and Pain Medicine (Jahr, Lurie) and Medical Pathology
(Tang, Louie, Kost), University of California–Davis Medical Center, Sacramento, Calif; and the University of Pennsylvania, School of Veterinary
Medicine, Department of Clinical Studies (Driessen), New Bolton Center, Kennett Square, Pa. Corresponding author: Bernd Driessen, DVM, PhD,
University of Pennsylvania, School of Veterinary Medicine, Department of Clinical Studies, New Bolton Center, 382 W Street Road, Kennett Square,
PA 19348 ([email protected]).
Vol. 30 / No. 1 / 2001
Veterinary Clinical Pathology
Page 39
Validation of Co-Oximetry for HBOC-Containing Blood
dard oxyhemoglobin dissociation curve usually derived
from human blood with normal shape and position (P50)
of the curve.18 In samples with low PO2 (for example
mixed venous blood) this calculation has been shown to
be associated with significant error.19-21 Therefore, cooximetry is recommended for purposes of systemic oxygen uptake computations.21
The use of xenogeneic, that is, heterogeneous, Hb
such as Hb-200 adds another dimension to the complexity of interpreting blood gas and co-oximetry data.
As a result of the glutaraldehyde-polymerization process, Hb-200 and related blood substitutes (HBOC-201)
have a different O2 dissociation curve with a P50 that is
right shifted to 34-38 mm Hg5,8 as compared to the P50 of
2822 and 26 mm Hg8 for native canine and human Hb,
respectively. Moreover, blood gas analyzers and cooximeters usually compute oxygen content (O2ct) based
on the oxygen-binding capacity (Hüfner factor: 1.39
mL/g)23 of human Hb. For these reasons, it is necessary
to use different equations for calculations of Hb saturation and total blood O2ct when blood that contains xenogeneic Hb is analyzed.21,24
Co-oximetry, a photometric method operating with
light in the wavelength band of 500-670 nm, has its own
limitation when a mixture of 2 Hb species differing in
their light absorption characteristics is analyzed.
Photometric measurements of SO2 and subsequent computation of blood O2ct become particularly questionable
when xenogeneic Hb is used in patients as a blood-substituting oxygen carrier.
Although certain laboratory tests were validated for
the use of HBOC-201, in all those tests HBOC-201 was
added to plasma instead of to whole blood.19,20,25 Thus,
the potential for interactions of stroma-free Hb with cellular components, which might have influenced certain
test assays, was not addressed in those studies. No independent reports are available on validation of photometry-based measurements of SO2 in whole blood containing Hb-200 in various concentrations.
This study was designed to validate the NOVA CoOximeter (Nova Biomedical Corporation, Waltham,
Mass, USA) for SO2 measurements under conditions
similar to the clinical application of HBOCs. Canine
blood was used in these in vitro experiments.
Materials and Methods
Instrumentation
Concentrations of Hb fractions and SO2 were measured
with the NOVA CO-Oximeter, which computes total
O2ct based on measurements. Co-oximetry is a photometric method used for the determination of Hb oxygen
saturation and is based on the phenomenon that light
Page 40
absorption of oxygenated Hb (oxy-Hb) is different from
that of deoxygenated Hb (deoxy-Hb). Typically, absorption spectra are measured at different wavelengths in
the 500- to 670-nm band (eg, 535.0, 585.2, 594.5, and 626.6
nm). Blood containing foreign stroma-free Hb might
have oxy-Hb absorption peaks different from that of
native blood.24
Quantitative measurements of O2ct were performed
with an oxygen-specific electrode, also called a fuel cell
(LEXO2CON-K, Hospex Fiberoptics, Chestnut Hill,
Mass, USA). This instrument measures the total of both
dissolved and bound O2 in blood, molecule by molecule,
and is therefore accurate in all ranges, with all blood of
all species, regardless of whether or not the blood sample contains any stroma-free Hb in addition to red blood
cell Hb. For this reason, the oxygen fuel cell is considered the gold standard technique for the quantification
of O2ct.
Two IL tonometers (Model 237, Instrumentation
Laboratory, Lexington, Mass, USA) were used to equilibrate the PO2 and PCO2 in blood samples with a gas mixture of O2, CO2, and nitrogen (balance) in a tank. To do
so, a cuvette is filled with the blood sample and then set
into an equilibration chamber. The equilibration chamber is surrounded by a thermostat-controlled water bath
that has a temperature preset by the operator. During
the equilibration process, prewarmed humidified gas
flows through the chamber. At the same time the
cuvette undergoes a constant 2-stage stirring cycle, creating a thin film of blood in the cuvette that allows more
blood to be exposed to the gas. The tonometry gases are
certified standards with a guaranteed error of ± 0.03%.
Sample preparation
For the first set of experiments, blood from 6 healthy and
transmittable disease–free donor dogs (mixed-breed
dogs; 3-8 years of age; mean [± SD] body weight 30 ± 3
kg; 4 males, 2 females) was obtained from the animal
blood bank of the University of California–Davis,
Veterinary Medical Teaching Hospital. Both the blood
donor program and the use of these animals as blood
donors was approved by the Campus Animal Care and
Use Committee and the U.S. Department of Agriculture.
All animals were treated at all times in compliance with
the Guide for the Care of Laboratory Animals (National
Institutes of Health publication 86-23, revised 1985). The
blood from these 6 donors had been stored for no longer
than 3 weeks from the day of withdrawal, and had originally been collected in plastic containers containing citrate-phosphate-dextrose (CPD; 0.15 mL of solution per
mL of whole blood). The hematocrit of the stored blood
specimens, as measured by capillary tube centrifugation, ranged from 34% to 37%. The corresponding mean
Veterinary Clinical Pathology
Vol. 30 / No. 1 / 2001
(± SD) Hb concentration was 11.8 ± 0.2 g/dL.
For the second series of experiments, small volumes
(12 mL) of arterial and venous fresh whole blood were
withdrawn via the dorsal pedal artery and cephalic vein,
respectively, from the 6 blood donor dogs on days when
the animals were scheduled for blood donation. The
blood samples were collected in heparinized syringes,
air sealed, stored on ice (3-4°C), and immediately taken
to the laboratory for processing and measurements.
They had an average (± SD) Hb concentration of 14.3 ±
2.7 g/dL.
Hemoglobin glutamer-200 was added in vitro under
anaerobic conditions to aliquots of the canine donor
blood to achieve extracellular (plasma) Hb concentrations ranging from 1.62 to 9.75 g/dL. Samples with extracellular Hb concentrations of 1.62 and 3.25 g/dL were
used for tonometry. Samples with extracellular Hb concentrations of 1.62, 3.25, 6.50, and 9.75 g/dL were used for
O2ct measurements. Canine donor blood and Hb-200
(13.0 g/dL) were used as controls.
Experimental protocol
In the first set of experiments, the target PO2 levels were
40, 80, 160, and 400 mm Hg. Each tonometer was connected to a gas tank containing O2 in concentrations of
5.62%, 11.25%, 22.48%, or 56.05%. Each tank contained
5.62% CO2 and nitrogen (N2) for balance.The tonometry
order was randomized. From each of the 6 donor blood
samples, 2 mL of blood were placed into a cuvette, and
after a 15-minute equilibration period at 37°C, an
aliquot of blood was drawn into a syringe anaerobically.
The sample was immediately analyzed in the NOVA
CO-Oximeter at a temperature of 37 ± 1°C.This test was
performed 3 times with a 1-minute interval between
each sample. The atmospheric pressure on the day of
the experiment was recorded with a barometer in the
laboratory. The actual PO2 in the samples was calculated
as:
PO2 = (Patm – PH20) % O2 ,
where Patm is the atmospheric pressure, PH2O is the water
vapor pressure at 37°C, and %O2 is the oxygen concentration of the gas mixture in the tonometry tank.
Oxygen saturations were then calculated for each
Hb species (canine and bovine) separately on the basis
of the actual PO2 obtained, using the standard oxy-Hb
dissociation curve as derived from the Kelman equation26-29 for canine Hb, and the corresponding curve
reported for the pure bovine HBOC solution for Hb200.7 This computation procedure included temperature
correction to 37°C as previously described by Nunn.26
Total SO2 of the blood sample was calculated based on
the concentration of each Hb species in the sample. Red
Vol. 30 / No. 1 / 2001
Measured O2 saturation (%)
Jahr, Driessen, Lurie, Tang, Louie, Kost
r = 0.98
Calculated O2 saturation (%)
Figure 1. Correlation between measured and calculated oxygen
saturation (SO2) values in native canine blood and blood containing hemoglobin glutamer-200 (bovine; Hb-200). Target blood P02
was reached by tonometry in aliquots of native canine blood and
canine blood mixed with Hb-200 at 1.62 or 3.25 g/dL. Total SO2
was then measured with the NOVA CO-Oximeter or calculated for
each hemoglobin species based on the actual P02 of the sample.
Data points are the means of 6 samples measured in triplicate. The
Pearson correlation coefficient is given.
blood cell Hb concentration was determined in each
tested sample.
In the second set of experiments, co-oximetry and
quantitative measurements of O2ct with the oxygenspecific electrode were performed in triplicate on samples of fresh whole blood immediately after withdrawal.
Samples of arterial, venous, and mixed (50% arterial,
50% venous) whole blood from each of the 6 dogs were
used to measure O2ct at 3 different levels of SO2. To
obtain similar “arterial,” “venous,” and mixed samples
with pure Hb-200 solution, Hb-200 solution was exposed by tonometry to oxygen tensions measured in
corresponding arterial and venous whole blood samples
from the donor dogs, and a mixed sample was prepared
as described previously.
Based on co-oximetry data, total oxygen content
was calculated for each sample using the following
equation:
O2ct = (SO2)(1.32bHb + 1.36cHb) + PO2 0.003,
where SO2 is the Hb oxygen saturation, bHb is the
bovine Hb concentration, cHb is the canine Hb concentration, and the constants 1.32 and 1.36 are the theoretical O2 binding capacities (Hüfner factors [mL/g]) of 1 g
of Hb-200 and native canine Hb with molecular weights
of 68,000 and 64,458 d, respectively.28-32
Veterinary Clinical Pathology
Page 41
Validation of Co-Oximetry for HBOC-Containing Blood
Table 1. Calculated and measured oxygen saturation (SO2; in %) in stored canine blood (native blood) and canine blood containing hemoglobin glutamer-200 (bovine; Hb-200). Target PO2 was adjusted by tonometry, and SO2 was measured by the NOVA-CO-Oximeter or was
calculated for each hemoglobin species based on actual PO2 values. Data are the mean ± SD of 6 samples measured in triplicate.
Actual PO2
(mm Hg)
Concentration of Hb-200 in the Sample (g/dL)
1.62
0 (native blood)
3.25
SO2
Calculated
Mean SO2
Measured
Difference
(%)
SO2
Calculated
Mean SO2
Measured
Difference
(%)
SO2
Calculated
Mean SO2 Difference
Measured
(%)
39.9
76.0
74.3 ± 0.2
2
70.4
66.6 ± 0.2
5
56.1
54.1 ± 0.3
4
80.0
95.4
92.8 ± 0.2
3
93.6
88.6 ± 0.3
5
89.1
85.6 ± 0.5
4
159.9
99.3
98.3 ± 0.3
1
98.9
93.9 ± 0.4
5
98.1
95.4 ± 0.2
3
398.8
99.9
99.3 ± 0.2
1
99.9
99.4 ± 0.3
1
99.8
97.6 ± 0.1
2
Measured O2 content (mL/dL)
tween whole blood and blood mixtures with Hb-200
were made with a 1-way ANOVA. A value of P<.05 was
always taken to be statistically significant.
Results
r = 0.98
Calculated O2 content (mL/dL)
Figure 2. Correlation between measured and calculated total oxygen content (O2ct) in native canine blood and blood containing
hemoglobin glutamer-200 (bovine; Hb-200). In aliquots of canine
fresh whole arterial, venous, and mixed (50% arterial, 50%
venous) blood; canine arterial, venous, and mixed blood mixed
with Hb-200 at 1.62, 3.25, 6.50, or 9.75 g/dL; and in pure Hb-200
solution (bovine hemoglobin at 13.0 g/dL) adjusted to corresponding P02 values, O2ct was measured with an oxygen-specific electrode or calculated based on oxygen saturation and hemoglobin
measurements with the NOVA CO-Oximeter. Data points are the
means of 6 samples measured in triplicate. The Pearson correlation coefficient is given.
Statistical analysis
All values are given as mean ± SD. The Pearson coefficient of correlation was used for comparison between
calculated and measured values. The hypothesis of zero
bias was examined by paired t-test.33 Comparisons be-
Page 42
The SO2 values measured with the NOVA CO-Oximeter
in samples with known PO2 demonstrated strong correlation with the calculated SO2 (Figure 1). Changing the
concentration of Hb-200 did not significantly affect the
results (Table 1). The difference between measured and
calculated SO2 values did not exceed 5% and did not significantly change after adding Hb-200 in concentrations
of 1.62 and 3.25 g/dL to the canine blood. However, the
comparison of calculated and measured SO2 values of
the native blood group with those of the test groups containing either 1.62 or 3.25 g/dL Hb-200 revealed a significant difference. Increasing the concentration of Hb-200
significantly increased in a dose-dependent manner the
difference in both calculated and measured SO2 values
between whole blood and the mixtures of whole blood
with Hb-200, provided the PO2 was adjusted to O2 tensions of only 80.0 and 39.9 mm Hg (P< .05).
In samples with known PO2, O2ct values, when calculated based on co-oximetry results, demonstrated
strong correlation with actual O2ct values measured by
the oxygen-specific electrode (Figure 2). Calculated O2ct
of the blood was in the same 5% difference range compared to measurements with the oxygen-specific electrode. The results did not change with different oxygenation of the sample. The difference between the 2
methods also did not change significantly when the concentration of Hb-200 was increased from 1.62 to 9.75
g/dL, or when O2ct was measured in native canine blood
and pure Hb-200 solution (13.0 g/dL; Table 2). The overall correlation between the 2 methods (r = .98) was statistically significant.
Veterinary Clinical Pathology
Vol. 30 / No. 1 / 2001
Vol. 30 / No. 1 / 2001
Veterinary Clinical Pathology
2
17.1±0.3
16.8
5
19.8±0.1
20.9
2
22.0±0.3
21.5
*D indicates the percentage difference between calculated and measured values (%).
4
21.8±0.4
21.0
4
20.6±0.3
21.4
5
20.6
Arterial
19.5±0.2
4
11.6±0.1
11.2
4
13.2±0.2
13.8
5
15.0±0.3
14.3
3
14.4±0.3
13.9
4
13.6±0.1
14.2
5
13.6
Mixed
12.9±0.1
5
8.1±0.1
7.7
5
9.1±0.1
9.6
4
9.5±0.1
9.9
4
9.3±0.2
9.7
5
10.3±0.2
9.8
5
9.9±0.2
9.4
O2ct-C
D*
O2ct-M
O2ct-C
D*
O2ct-M
O2ct-C
D*
O2ct-M
O2ct-C
D*
O2ct-M
O2ct-C
0
(native blood)
Venous
D*
O2ct-M
O2ct-C
O2ct-M
D*
13.0
(pure Hb-200)
9.75
6.50
3.25
1.62
Concentration of Hb-200 in the Sample (g/dL)
Oxygen saturation of blood is one of the most important
parameters used in anesthesia and critical care monitoring of patients,34 as well as in a wide variety of experimental studies.5 In the hospital setting, co-oximetry is a
commonly used method for determining SO2 but has
not been validated for measurement of SO2 in blood
containing HBOC solutions such as Hb-200.
In the present in vitro study, we validated co-oximetry data by testing samples of canine blood mixed with
Hb-200 in different concentrations. First, samples with
known PO2 were prepared with tonometers and tested
with the co-oximeter. In a separate set of experiments,
O2ct was directly measured and then compared with
values calculated based on Hb fractions; SO2 was measured with the NOVA CO-Oximeter.
Oxygen saturation measured by co-oximetry represents the percentage of oxy-Hb, expressed as a fraction
of total Hb (oxy-Hb plus deoxy-Hb). Serum containing
polymerized bovine Hb solution, whether oxygen saturated or not, has been demonstrated to display absorption peaks at 415, 540, and 576 nm, which are characteristic of oxy-Hb,24 and, therefore, may interfere with cooximetry accuracy. In contrast to native canine Hb,
which is virtually fully O2-saturated under room air conditions (21% O2 ; arterial PO2 100 mm Hg) and body
temperature, bovine HBOC solutions such as Hb-200
and HBOC-201 are only 83-85% oxygen saturated under
the same conditions because of their higher P50 of 34-38
mm Hg.7,8 Hence, at PO2 values of <100 mm Hg, the light
absorption by a nonoxygenated subfraction of the
bovine Hb solution was expected to produce falsely
high co-oximetry readings of SO2 compared to calculated values. In contrast to this expectation, we obtained
slightly lower readings with co-oximetry, which were
still within a 5% difference between calculated and
measured values (Table 1). Parameters such as pH, PCO2,
and electrolyte content, which were not measured in
these experiments, may have been slightly different
from physiologic levels in mixed whole blood/Hb-200
samples and were not taken into account in calculating
SO2. Other factors, unknown to us, also may have contributed to the minimal difference between measured
and calculated SO2 values.
However, the higher P50 of Hb-200 might explain
why at oxygen tensions < 100 mm Hg (39.9 and 80.0 mm
Hg), both calculated and measured SO2 values were significantly lower in samples containing mixtures of canine
blood and Hb-200 than in native canine blood samples.
This difference became particularly obvious at the higher
Hb-200 concentration, supporting this assumption.
For the reasons mentioned above, we predicted O2ct
to be higher when calculated from co-oximetry SO2 data
Sample
Type
Discussion
Table 2. Calculated oxygen content (O2ct-C, mL/dL; based on oxygen saturation and hemoglobin measurements with the NOVA-CO-Oximeter) and measured oxygen content
(O2ct-M, mL/dL; directly measured by oxygen-specific electrode) in native canine fresh whole blood, canine fresh whole blood containing hemoglobin glutamer-200 (bovine; Hb200), and pure Hb-200 solution. Data are the mean ± SD of 6 samples measured in triplicate.
Jahr, Driessen, Lurie, Tang, Louie, Kost
Page 43
Validation of Co-Oximetry for HBOC-Containing Blood
than when measured with the oxygen-specific electrode. In our experiment, the difference between calculated and measured values for O2ct did not exceed 5%.
A statistically significant correlation was found between
measured and calculated values with co-oximetry data.
Concentrations of Hb-200 in blood plasma can
reach 3.25 g/dL when approximately 25% of the circulating blood volume is substituted, which is considered a
clinically relevant target.5 In our tests, concentrations of
1.62, 3.25, 6.50, and even 9.75 g/L did not cause significant changes in co-oximetry results and did not increase
the discrepancy between calculated and measured
parameters.
In conclusion, the strong correlation between measured and calculated values allowed us to consider cooximetry an accurate technique for measurement of SO2
in canine blood after infusion of Hb-based oxygen carriers such as Hb-200. ◊
Acknowledgments
The authors thank Joan Bullock for assistance with the NOVA COOximeter, and the Animal Blood Bank of the University of
California–Davis, Veterinary Medical Teaching Hospital for providing blood from their canine blood donors.
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