Anesth Analg 2002;95:870-873
© 2002 International Anesthesia Research Society
CARDIOVASCULAR ANESTHESIA
The Novel HemoCue® Plasma/Low Hemoglobin System Accurately Measures Small Concentrations of Three Different Hemoglobin-Based Oxygen Carriers in Plasma: Hemoglobin Glutamer-200 (Bovine) (Oxyglobin®), Hemoglobin Glutamer-250 (Bovine) (Hemopure®), and Hemoglobin-Raffimer (HemolinkTM)
Fedor Lurie, MD PhD*,
Jonathan S. Jahr, MD
, and
Bernd Driessen, DVM PhD
*Department of Surgery, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii; Department of Anesthesiology, University of California-Los Angeles School of Medicine, Los Angeles, California, and Department of Anesthesiology, Charles R. Drew University of Medicine and Science, Martin Luther King, Jr./Drew Medical Center, Los Angeles, California; and Department of Clinical Studies, University of Pennsylvania, School of Veterinary Medicine, Philadelphia, Pennsylvania
Address correspondence and reprint requests to J. S. Jahr, MD, UCLA Anesthesiology, Box 951778, Los Angeles, CA 90095-1778. Address e-mail to jsjahr{at}mednet.ucla.edu
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Abstract
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The accuracy of the HemoCue® Plasma/Low Hemoglobin System was validated in vitr. with low levels of hemoglobin-based oxygen carriers (HBOCs). Repeated measurements were performed on 50 samples of canine plasma, each mixed with three different HBOCs at varying small concentrations (a total of 150 samples), by using plasma samples without HBOCs as controls. Two technicians performed the measurements and randomly tested each sample 10 times. The results were analyzed for correlation, and analysis of variance was used to evaluate statistical significance, with a P value of 
0.05 considered significant. Hemoglobin concentrations determined with the bedside photometer were not significantly different from known values of hemoglobin concentration in the samples. There was no significant difference between values obtained by two independent observers for the same samples. This was true for all three tested HBOCs and for all tested concentrations. The mean bias of the measurement expressed as a percentage of sample concentration was 0.1% for hemoglobin glutamer-200 (bovine), 0.58% for hemoglobin glutamer-250 (bovine), and 0.19% for hemoglobin-raffimer. The mean error was <8% for all three HBOCs. Both intraobserver and interobserver reliabilities were high and statistically significant. The HemoCue® Plasma/Low Hemoglobin System is a reliable instrument for detecting and measuring small concentrations of three different HBOCs in plasma.
IMPLICATIONS: This study evaluated a new bedside blood-measuring device for low levels and found that it rapidly measured low levels accurately for three blood substitutes.
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Introduction
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The potential clinical use of hemoglobin-based oxygen carriers (HBOCs) introduces additional complexity in measuring plasma levels of hemoglobin (Hb) by presenting extracellular, allogeneic Hb into the circulation. The plasma Hb concentration is an important variable for detecting hemolytic conditions, including those during transfusion therapy in patients with major blood loss, creating a need for an accurate point-of-care monitor for low plasma Hb. Therefore, accurate measurement of the circulating HBOCs after infusion becomes an important issue, indicating a need for such a device.
A novel point-of-care hemoglobinometer for measuring small concentrations of plasma Hb, the HemoCue® (Plasma/Low Hemoglobin System; Aktiebolaget Leo Diagnostics, Helsigborg, Sweden), provides rapid and convenient assessment of the Hb concentration. This device uses a chemically pretreated cuvette for the blood sample. The reagents deposited on the inner wall of the cuvette lyse the red cells and convert Hb into azide methemoglobin. The Hb concentration is calculated by using spectrophotometric analysis with absorbance peaks at 565 and 880 nm. The manufacturer claims the accuracy of the device to be within ±0.03 g/dL (1). However, this system has never been tested for use with small concentrations of HBOCs. In a recent study, we evaluated the currently available HemoCue® B-Hemoglobin Photometer with three HBOCs (2).
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Methods
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In the laboratory, repeated measurements were conducted on 50 samples of canine plasma mixed with each of the HBOCs in 5 different concentrations (a total of 150 samples). The canine plasma was derived from the remainder of a healthy single dog’s plasma sample that was drawn for other purposes. For Hb glutamer-200 (bovine) and Hb glutamer-250 (bovine), the concentrations tested were 1.3, 0.65, 0.26, 0.13, and 0.065 g/dL; for Hb-raffimer they were 1.00, 0.5, 0.25, 0.1, and 0.05 g/dL. Plasma samples containing no HBOC were used as controls. Samples were prepared by serial dilutions. The Hb concentration in the HBOC stock solution was determined by using both an automated laboratory analyzer (Coulter STCS; Coulter Corp., Hialeah, FL) and a HemoCue® B-Hemoglobin Photometer (2). Two experienced independent laboratory technicians were blinded to sample number and performed the photometer measurements. Each operator randomly tested each dilution sample 10 times. The results were analyzed for correlation, and analysis of variance was used to evaluate statistical significance, with a P value of <0.05 considered significant. The bias was defined as a mean of difference and the error as a standard deviation of the difference between the Hb concentration in the sample and the measured value. The intra- and interobserver reliability were calculated Pearson coefficients of correlation. The coefficient of repeatability was calculated as defined by the British Standards Institution (3). A Bland and Altman analysis was not used because the instrument was tested against known (titrated) concentrations and not compared with another instrument (4,5). No current laboratory equipment is available to measure very small Hb concentrations as a "gold standard."
The three HBOCs used in this study were obtained as follows: the Hb glutamer-200 (bovine) (Oxyglobin®; Biopure Corp., Cambridge, MA) was purchased through the veterinary pharmacy at the University of California-Davis School of Veterinary Medicine. The Hb-raffimer (HemolinkTM; Hemosol, Inc., Toronto, Ontario, Canada) was donated by Hemosol, Inc., and the Hb glutamer-250 (Hemopure®; Biopure Corp.) was donated by Biopure Corp. The colloid osmotic pressures were measured for each of the three products and were within the standard range reported in the literature (6–8) (Table 1); baseline Hb values were measured and were accurate on the basis of the reported Hb content of the products (Table 1).
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Table 1. Physical and Chemical Properties of Hemoglobin Glutamer-200, Hemoglobin Glutamer-250, and Hemoglobin-Raffimer
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Results
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Hb concentrations determined with the HemoCue® Plasma/Low Hemoglobin System photometer were not significantly different from values of Hb concentration in the samples (Table 2). There was also no significant difference between values obtained by two independent observers for the same samples. This was true for all three tested HBOCs and for all tested concentrations.
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Table 2. Hemoglobin Concentration Values Obtained by Two Independent Observers Using the HemoCue® Plasma/Low Hemoglobin System
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The mean bias of the measurement expressed as a percentage of sample concentration was 0.1% for Hb glutamer-200 (bovine), 0.58% for Hb glutamer-250 (bovine), and 0.19% for Hb-raffimer. The mean error was <8% for all three of the tested HBOCs.
Both intraobserver and interobserver reliabilities were high and statistically significant (Table 3). Because each of the observers performed 10 measurements for each of the concentrations of each of the HBOCs, repeatability coefficients were calculated (Table 4), which showed very high repeatability of the measured values.
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Discussion
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This study assessed the accuracy of the photometric measurement of the Hb concentration by using the HemoCue® and three different HBOCs: Hb glutamer-200 (bovine) (Oxyglobin®), Hb glutamer-250 (bovine) (Hemopure®), and Hb-raffimer (HemolinkTM) in varying small concentrations mixed with canine plasma. One of these HBOCs (Hb glutamer-200) is Food and Drug Administration (FDA)-approved for veterinary use and is indicated for canine anemia, Hb glutamer-250 and Hb-raffimer have currently completed or are undergoing FDA Phase III trials in the United States, and Hb glutamer-250 is approved for human use in South Africa. The published chemical and physical properties of the three HBOCs are shown in Table 1 (2).
The clinical accuracy and reliability of the currently available HemoCue® B-Hemoglobin Photometer have been confirmed for larger concentrations of Hb (2,9–11). Some technical aspects of the technology, however, have been emphasized in two articles (10,12). The dependence of the results on the use of appropriate technique leads to some degree of intraobserver variability (9,10,12). The new HemoCue® Plasma/Low Hemoglobin System was designed to measure smaller concentrations of Hb. It uses different cuvette sizes with a larger volume (20 µL) and an increased distance between the walls of the optical window (0.40 mm), compared with 10 µL and 0.13 mm, respectively, for the B-Hemoglobin Photometer (1,11). The proportions of sodium deoxycholate and sodium azide in the mixture of reagents have been increased by 4% each compared with the B-Hemoglobin Photometer (1,11). With these and other changes, the measurement range of the Plasma/Low Hemoglobin System is 0.03 to 3.0 g/dL (1).
The results of measurements with the B-Hemoglobin Photometer become particularly important when concentrations of Hb in plasma are measured that approach the limit of accuracy for the instrument (1). The use of HBOCs makes it clinically important to validate the new HemoCue® Photometer for measurement of plasma Hb. Moreover, some of the HBOCs under development are heterogeneous Hbs or chemically altered Hbs; therefore, they have optical properties different from those of normal human erythrocytic Hb. These possibly can alter the accuracy of photometric measurements (6,12,13).
In a recently published study, we evaluated the currently available HemoCue® B-Hemoglobin Photometer with three HBOCs, the same as those studied in the present study (2). We found that the B-Hemoglobin Photometer accurately determined the concentration of the three HBOC solutions dissolved in canine plasma. It was important to assess the new photometer, because it was designed especially for small concentrations of Hb that are clinically relevant to patients treated with HBOCs (14,15). Both HemoCue® studies used canine plasma, which is similar to human plasma, and the results would not be expected to be altered if human plasma were used (2).
The doses of HBOCs tested in this study are relevant in humans in that concentrations of 0.5 g/dL have been shown to deliver oxygen to tissue (16), and athletes have been illegally administered small concentrations of HBOCs to improve exercise tolerance (17,18). The machine is novel in that it has a lower limit of accuracy (0.03 g/dL), and we tested levels down to 0.05 and 0.065 g/dL, indicating that the machine is validated as it approaches the lower range of claimed accuracy (1).
Additionally, one HBOC has been evaluated to assess circulating blood volume accurately by means of an indicator dilution technique, where the HBOC serves as the indicator (19). The accurate measurement of low plasma levels of HBOCs is crucial, not only in cases in which they are used for the determination of the circulating plasma volume, but also in those in which one intends to determine dose-response relationships or to observe the pharmacokinetic profile of those Hb solutions.
A recent study evaluated eight cooximeters in five HBOCs (20). The only HBOC in clinical Phase III trials studied was HemolinkTM. The other HBOCs are not in clinical trials, except for Oxyglobin®, which is approved for veterinary use. The results of the study by Ali et al. (20) are informative in that, in general, the presence of HBOCs is less accurate but yields clinically useful measurements of oxy-, deoxy-, carboxy-, and methemoglobin.
This study demonstrates that in the clinically relevant range of small plasma concentrations of three different HBOCs, the Hb concentrations determined by use of the HemoCue® Plasma/Low Hemoglobin System are reliable and accurate. The difference between two independent observers never reached the level of statistical or clinical significance. Both interobserver reliability and intraobserver repeatability were sufficiently high. On the basis of this in vitr. experiment, we conclude that the B-Hemoglobin Photometer is an appropriate device for rapid Hb concentration measurements (approximately one minute) when HBOCs are infused, including for very small Hb concentrations.
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Acknowledgments
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Funding for the study was from departmental sources.
The authors are grateful to the HemoCue Corporation for providing the HemoCue® Plasma/Low Hemoglobin System for evaluation and to Hemosol, Inc., and Biopure Corp. for donating HemolinkTM and Hemopure® to test in laboratory equipment. Dr. Jahr was the principle investigator in two Biopure and one Hemosol Phase III trials and is on the Biopure Speakers Bureau.
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Footnotes
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Presented in part at the annual meeting of the American Society of Anesthesiologists, New Orleans, LA, October, 2001.
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References
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- HemoCue® (plasma/low hemoglobin system): operating manual. Helsigborg, Sweden: Aktiebolaget Leo Diagnostics, 2000.
- Jahr JS, Lurie F, Driessen D, et al. The HemoCue®, a point of care B-hemoglobin photometer, accurately measures hemoglobin levels when mixed in vitro with canine plasma and three hemoglobin-based oxygen carriers (HBOC). Can J Anaesth 2002; 49: 243–8.[Abstract/Free Full Text]
- British Standards Institution. Precision of test methods. I. Guide for the determination and reproducibility for a standard test method (BS 5497, part 1). London: British Standards Institution, 1979.
- Bland JM, Altman DG. Measuring agreement in method comparison studies. Stat Methods Med Res 1999; 8: 135–60.[Abstract/Free Full Text]
- Mantha S, Roizen MF, Fleisher LA, et al. Comparing methods of clinical measurement: reporting standards for Bland and Altman analysis. Anesth Analg 2000; 90: 593–602.[Abstract/Free Full Text]
- Jahr JS, Lurie F, Driessen B, et al. Validation of the oxygen saturation measurements in a canine model of hemoglobin-based oxygen carrier (HBOC) infusion. Clin Lab Sci 2000; 13: 173–9.
- Kasper SM, Walter M, Grüne F, et al. Effects of a hemoglobin-based oxygen carrier (HBOC-201) on hemodynamics and oxygen transport in patients undergoing preoperative hemodilution for elective abdominal aortic surgery. Anesth Analg 1996; 83: 921–7.[Abstract]
- Manning JE, Katz LM, Brownstein CC, et al. Bovine hemoglobin-based oxygen carrier (HBOC-201) for resuscitation of uncontrolled, exsanguinating liver injury in swine: Carolina Resuscitation Research Group. Shock 2000; 13: 152–9.[ISI][Medline]
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- Jahr JS, Lurie F, Gosselin R, et al. Effects of a hemoglobin-based oxygen carrier (HBOC-201) on coagulation testing. Clin Lab Sci 2000; 13: 210–4.[Medline]
- Driessen B, Jahr JS, Lurie F, et al. Effects of haemoglobin-based oxygen carrier hemoglobin glutamer-200 (bovine) on intestinal perfusion and oxygenation in a canine hypovolaemia model. Br J Anaesth 2001; 86: 685–92.
- Standl T, Horn P, Wilhelm S, et al. Bovine hemoglobin is more potent than autologous red blood cells in restoring muscular tissue oxygenation after profound isovolaemic haemodilution in dogs. Can J Anaesth 1996; 43: 714–23.[Abstract/Free Full Text]
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- Ali AA, Ali GS, Steinke JW, Shepard AP. Cooximetry interference by hemoglobin-based blood substitutes. Anesth Analg 2001; 92: 863–9.[Abstract/Free Full Text]
Accepted for publication May 28, 2002.
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Abstract
Introduction
