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Hemoglobin-based oxygen carriers (HBOCs) are now undergoing extensive clinical trials to determine their therapeutic potential (1). HBOCs in plasma resemble massive hemoglobinemia and create a challenge for the chemistry laboratory to accurately measure analytes in their presence. HemolinkTM, like hemoglobin and other HBOCs, has been found to interfere with common laboratory test methodologies (2). Interference from HBOCs is caused by an increase in absorbance or by interference in certain chemical reactions (e.g., peroxidase-like activity) (3)(4). It is therefore necessary to measure affected serum analytes by alternative methods, utilize correction factors, or remove the interfering HBOC to obtain accurate results (5)(6)(7). Hemolink comprises a mixture of stabilized human hemoglobin molecules (64–600 kDa). Periodate-oxidized raffinose (o-raffinose) cross-links the ß-subunits at Lys-82 to form stabilized hemoglobin tetramers, and in addition intermolecularly cross-links tetramers through surface amino groups to form hemoglobin polymers.
Recently, a new product that has been reported to selectively bind and remove free hemoglobin from clinical samples has come onto the market. HemogloBindTM is an insoluble anionic polyelectrolyte manufactured by LigoChem (Fairfield, NJ). The application of HemogloBind for the removal of Hemolink and other HBOCs would reduce the potential for interference in laboratory methods. The objective of this study was to evaluate the efficiency of HemogloBind in removing Hemolink from serum. Parallel experiments were also done with highly purified human hemoglobin (HemAzeroTM).
To pooled human serum we added Hemolink or hemoglobin, 2–20 g/L. Samples were treated with HemogloBind according to the manufacturer's standard protocol. In brief, HemogloBind was resuspended by vortex-mixing and 100 µL was added to 200 µL of the supplemented serum samples. The samples were mixed end-over-end (Lab-Quake rotator; Barnstead/Thermolyne, IA) for 15 min, allowed to sit for 1 min, and then centrifuged (3000g for 10 min). The supernatant was carefully removed and analyzed for Hemolink or hemoglobin and total protein. All values were multiplied by 1.45 to correct for dilution with HemogloBind. A modified Drabkin hemoglobin assay (8) was used to quantify unbound Hemolink and hemoglobin. A calibration curve (50–1000 mg/L) was constructed with Sigma's hemoglobin standard (no. 525–18) diluted with Sigma's Drabkin's solution (no. 525A). Aliquots of supernatant were mixed with Drabkin's solution (final volume 200 µL) and incubated for 15 min. Absorbance was measured at 540 nm with a THERMOmax microtiter plate reader (Molecular Devices Corp., Menlo Park, CA). The absorbance of Hemolink and hemoglobin was measured at 414 nm when the concentrations were below the assay range for the modified Drabkin assay. We estimated the total protein content in diluted samples (100-fold), using the Bio-Rad Labs. protein assay and a bovine serum albumin standard. Bio-Rad's microtiter plate protocol was used and the absorbance at 600 nm was read with the THERMOmax microtiter plate reader. Hemolink up to 0.05 g/L did not significantly interfere in this assay. Serum analytes were measured with the Vitros 750 analyzer (Johnson and Johnson Clinical Diagnostics): amylase, alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, bilirubin, creatine kinase, creatinine, glucose, lactate dehydrogenase, lipase, and urea.
HemogloBind was unable to completely remove Hemolink or hemoglobin at
any concentration tested in a serum matrix (Fig. 1
). At all concentrations tested, the relative percentage of
Hemolink or hemoglobin removed remained constant (24.6% ± 2.6% and
76.4% ± 4.5%, respectively). Consequently, the absolute amount of
Hemolink or hemoglobin removed increased with increasing concentration
applied. The threefold difference in the relative amount of Hemolink
removed in comparison with hemoglobin in serum could be accounted for
by the molecular mass distribution of Hemolink. Two Hemolink fractions,
64 kDa and >64 kDa, were obtained by gel-filtration chromatography.
The 64-kDa Hemolink fraction was removed to a greater extent than
the >64-kDa fraction (by about fourfold) under standard conditions.
In 10 mmol/L phosphate buffer, pH 7.2, however, the HemogloBind removal
of Hemolink or hemoglobin differed from its effectiveness in serum.
HemogloBind completely removed at least 20 g/L of hemoglobin and up to
6 g/L of Hemolink, in contrast to their fractional removal from serum.
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In contrast to our results, the manufacturer's performance claim for HemogloBind states that >90% of hemoglobin (4.5 g/L) added to neonatal serum can be removed. The discrepancy may result from the presence of haptoglobin in our adult serum samples, which is absent in neonatal serum samples (9). Haptoglobin would produce haptoglobin–hemoglobin complexes that may not be removed by HemogloBind (probably because of the size selectivity of the resin).
Further studies were done to determine whether manipulation of the treatment conditions could increase the capacity of HemogloBind for Hemolink. Increasing volumes of HemogloBind (100–400 µL) were added to serum (200 µL) that already contained Hemolink (10 g/L). More Hemolink was removed with increasing volumes of HemogloBind, but total protein recovery decreased concomitantly. In fact, ~90% of Hemolink could be removed by using 250 µL of HemogloBind. The total protein recovered, however, was only 52%, and the serum dilution factor increased to 2.125. In contrast, when serum volumes were varied (200–500 µL of serum to 100 µL of HemogloBind), less Hemolink was removed and slightly more protein was recovered. Also, when diluted serum samples were tested (diluted as much as fourfold), more Hemolink was removed at lower dilutions, and more protein was recovered. These studies supported the manufacturer's optimal HemogloBind:serum volume ratio of 1:2. Alteration of this ratio leads to changes in the binding characteristics of HemogloBind.
We also studied the effect of two volumes of HemogloBind on the measured concentrations of various analytes. Samples containing Hemolink at 2 g/L were treated with either 100 or 150 µL of HemogloBind (ratios of 1:2 and 1.5:2, respectively), resulting in the removal of 20% and 54% of Hemolink, respectively. For the analytes listed previously, the average measured values (as the percent of expected values) were 87% ± 8% and 79% ± 15% for the 1:2 and 1.5:2 samples, respectively. A few of the analytes tested were not reportable—either because of a dilution effect, which resulted in values below the analyzer range (amylase and creatine kinase), or interference from the remaining Hemolink (bilirubin). Lipase activity was greatly decreased after HemogloBind treatment (51% and 18% recovered for the 100- and 150-µL HemogloBind samples, respectively).
The concentrations at which hemoglobin or HBOCs can be tolerated on clinical analyzers depend on the particular test method (4). HBOC concentrations in clinical samples could be as high as 50 g/L, and few methods are unaffected at this concentration (6)(10). Therefore, a significant reduction in HBOC concentration would be needed to avoid any interference. The product HemogloBind was effective in removing only a small percentage of Hemolink at any concentration tested and was unable to reduce the concentration sufficiently to allow accurate performance of many clinical tests.
Footnotes
Hemosol Inc., 115 Skyway Ave., Etobicoke, Ontario, Canada M9W 4Z4
References
The following articles in journals at HighWire Press have cited this article:
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  A. A. Ali, G. S. Ali, J. M. Steinke, and A. P. Shepherd Co-Oximetry Interference by Hemoglobin-Based Blood Substitutes Anesth. Analg., April 1, 2001; 92(4): 863 - 869. [Abstract] [Full Text] [PDF]
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 Z. Ma, T. G. Monk, L. T. Goodnough, A. McClellan, M. Gawryl, T. Clark, P. Moreira, P. E. Keipert, and M. G. Scott Effect of hemoglobin- and Perflubron-based oxygen carriers on common clinical laboratory tests Clin. Chem., September 1, 1997; 43(9): 1732 - 1737. [Abstract] [Full Text] [PDF]
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