Clin Chem Lab Med 2015; 53(9): e219–e222
Letter to the Editor Françoise Arnaud*, Donna G. Sieckmann, Richard M. McCarron and Paula F. Moon-Massat
Hemoglobin measurements in samples containing hemoglobin-based oxygen carriers DOI 10.1515/cclm-2015-0197 Received February 25, 2015; accepted February 26, 2015; previously published online April 16, 2015
Keywords: artificial blood; instrument validation.
To the Editor, Hemoglobin-based oxygen carriers (HBOC) offer a bridging solution for emergency rescue of civilian or military victims where blood products may not be available. HBOCs have shown benefits compared to current resuscitative fluids, such as crystalloids and colloids, have shown improved blood oxygenation [1–3] and demonstrated survival benefits in pre-clinical [4] and clinical studies [5]. Analyzing blood samples containing HBOC for monitoring clinical situations presents some challenges. As HBOCs are extracellular agents, the measurement of plasma hemoglobin (Hb), primarily a reflection of HBOC, is essential for assessing their concentration. Furthermore, Hb can transform into methemoglobin (MetHb), which cannot carry oxygen. In addition, the wide range of molecular structures and sizes of HBOCs may complicate these measurements depending on the methodology of the instrument. *Corresponding author: Françoise Arnaud, PhD, Hematology, Naval Medical Research Center, NeuroTrauma Department, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500, USA, Phone: +1 301 3197687, E-mail: [email protected]; NeuroTrauma Department, Operational and Undersea Medicine Directorate, Naval Medical Research Center, Silver Spring, MD, USA; and Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD, USA Richard M. McCarron: NeuroTrauma Department, Operational and Undersea Medicine Directorate, Naval Medical Research Center, Silver Spring, MD, USA; and Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD, USA Donna G. Sieckmann and Paula F. Moon-Massat: NeuroTrauma Department, Operational and Undersea Medicine Directorate, Naval Medical Research Center, Silver Spring, MD, USA
Among novel HBOCs MP4CO-NP (Sangart, San Diego, CA, USA) (human Hb) and Sanguinate-CO™ (Prolong Pharmaceuticals, South Plainfield, NJ, USA) (bovine Hb) are both 100 kDa PEGylated Hb tetramers [6, 7] while HBOC-201 [HbO2 Therapeutics, LLC (formerly OPK Biotech, Cambridge, MA, USA)] is a 250 kDa, glutaraldehyde-polymerized bovine Hb. M101 (Hemarina, Morlaix, France) is a 3600 kDa naturally-occurring extracellular Hb polymer derived from a marine invertebrate (Arenicola marina) [8] and OxyVita C (OXYVITA, New Windsor, NY, USA) is an extremely large, 17,000 kDa, zero-linked polymer of bovine Hb [9]. The standard transformation of Hb and MetHb with cyanides to their stable forms (cyan-hemoglobins) allows the measurement of Hb and MetHb but due to the potential toxicity of cyanide, alternative techniques have been developed. One of them, the ABL-735 (Radiometer, Copenhagen, Denmark) has accurately read HBOC-201 samples [4, 10], it is unclear if other instruments can accurately measure these parameters with other HBOCs. The goal of the current study was to evaluate the accuracy of a panel of analytical devices in quantitating Hb and the accuracy the ABL-735 in measuring MetHb from samples containing HBOCs. The ABL-735 uses 128 wavelengths to generate a matrix of absorption coefficients for each Hb form to calculate the concentration of total Hb. The point-of-care HemoCue device (Haemocue, Ängelholm, Sweden) converts the Hb iron from the ferrous to the ferric state to form azidmethemoglobin. Absorbance is read at 570 nm and 880 nm (for turbidity compensation). The Advia-120 (Siemens Healthcare, Erlangen, Germany) and the HemaVet (Drew Scientific, Irvine, CA, USA), use a conventional cyanide method to transform Hb into cyanmethemoglobin. The absorbance is measured at 546 nm (Advia-120) or 540 nm (HemaVet). The HBOCs evaluated were: HBOC-201, M101, MP4CONP, Sanguinate-CO™, and OxyVita C. The latter three contain carbon monoxide. Human blood was obtained from the Walter Reed National Military Medical Center blood bank to set up an internal reference.
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e220 Arnaud et al.: Hb and MetHb detection with blood substitutes The blood or HBOCs were diluted with saline and samples were run in quadruplicate. All the instruments measured Hb and only the ABL-735 measured MetHb. Once the reference was established on the Advia-120, the Hb concentration of blood and HBOC samples were obtained with the other instruments. The nominal Hb concentration was back-calculated with respect to the dilution. The average Hb was compared to the manufacturer’s reference using the Student’s t-test. The data are presented as mean ± standard deviation (Table 1). Internal reference: The Advia-120 exhibited a high precision and linearity for Hb resulting in a reference Hb concentration of 12.22 ± 0.11 g/dL. Both the ABL-735 and HemoCue demonstrated a good accuracy of blood Hb compared to the reference, whereas the accuracy of the HemaVet readings were significantly lower by 9%–18% (p
Letter to the Editor Françoise Arnaud*, Donna G. Sieckmann, Richard M. McCarron and Paula F. Moon-Massat
Hemoglobin measurements in samples containing hemoglobin-based oxygen carriers DOI 10.1515/cclm-2015-0197 Received February 25, 2015; accepted February 26, 2015; previously published online April 16, 2015
Keywords: artificial blood; instrument validation.
To the Editor, Hemoglobin-based oxygen carriers (HBOC) offer a bridging solution for emergency rescue of civilian or military victims where blood products may not be available. HBOCs have shown benefits compared to current resuscitative fluids, such as crystalloids and colloids, have shown improved blood oxygenation [1–3] and demonstrated survival benefits in pre-clinical [4] and clinical studies [5]. Analyzing blood samples containing HBOC for monitoring clinical situations presents some challenges. As HBOCs are extracellular agents, the measurement of plasma hemoglobin (Hb), primarily a reflection of HBOC, is essential for assessing their concentration. Furthermore, Hb can transform into methemoglobin (MetHb), which cannot carry oxygen. In addition, the wide range of molecular structures and sizes of HBOCs may complicate these measurements depending on the methodology of the instrument. *Corresponding author: Françoise Arnaud, PhD, Hematology, Naval Medical Research Center, NeuroTrauma Department, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500, USA, Phone: +1 301 3197687, E-mail: [email protected]; NeuroTrauma Department, Operational and Undersea Medicine Directorate, Naval Medical Research Center, Silver Spring, MD, USA; and Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD, USA Richard M. McCarron: NeuroTrauma Department, Operational and Undersea Medicine Directorate, Naval Medical Research Center, Silver Spring, MD, USA; and Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD, USA Donna G. Sieckmann and Paula F. Moon-Massat: NeuroTrauma Department, Operational and Undersea Medicine Directorate, Naval Medical Research Center, Silver Spring, MD, USA
Among novel HBOCs MP4CO-NP (Sangart, San Diego, CA, USA) (human Hb) and Sanguinate-CO™ (Prolong Pharmaceuticals, South Plainfield, NJ, USA) (bovine Hb) are both 100 kDa PEGylated Hb tetramers [6, 7] while HBOC-201 [HbO2 Therapeutics, LLC (formerly OPK Biotech, Cambridge, MA, USA)] is a 250 kDa, glutaraldehyde-polymerized bovine Hb. M101 (Hemarina, Morlaix, France) is a 3600 kDa naturally-occurring extracellular Hb polymer derived from a marine invertebrate (Arenicola marina) [8] and OxyVita C (OXYVITA, New Windsor, NY, USA) is an extremely large, 17,000 kDa, zero-linked polymer of bovine Hb [9]. The standard transformation of Hb and MetHb with cyanides to their stable forms (cyan-hemoglobins) allows the measurement of Hb and MetHb but due to the potential toxicity of cyanide, alternative techniques have been developed. One of them, the ABL-735 (Radiometer, Copenhagen, Denmark) has accurately read HBOC-201 samples [4, 10], it is unclear if other instruments can accurately measure these parameters with other HBOCs. The goal of the current study was to evaluate the accuracy of a panel of analytical devices in quantitating Hb and the accuracy the ABL-735 in measuring MetHb from samples containing HBOCs. The ABL-735 uses 128 wavelengths to generate a matrix of absorption coefficients for each Hb form to calculate the concentration of total Hb. The point-of-care HemoCue device (Haemocue, Ängelholm, Sweden) converts the Hb iron from the ferrous to the ferric state to form azidmethemoglobin. Absorbance is read at 570 nm and 880 nm (for turbidity compensation). The Advia-120 (Siemens Healthcare, Erlangen, Germany) and the HemaVet (Drew Scientific, Irvine, CA, USA), use a conventional cyanide method to transform Hb into cyanmethemoglobin. The absorbance is measured at 546 nm (Advia-120) or 540 nm (HemaVet). The HBOCs evaluated were: HBOC-201, M101, MP4CONP, Sanguinate-CO™, and OxyVita C. The latter three contain carbon monoxide. Human blood was obtained from the Walter Reed National Military Medical Center blood bank to set up an internal reference.
Brought to you by | McMaster University Authenticated Download Date | 9/28/15 9:06 AM
e220 Arnaud et al.: Hb and MetHb detection with blood substitutes The blood or HBOCs were diluted with saline and samples were run in quadruplicate. All the instruments measured Hb and only the ABL-735 measured MetHb. Once the reference was established on the Advia-120, the Hb concentration of blood and HBOC samples were obtained with the other instruments. The nominal Hb concentration was back-calculated with respect to the dilution. The average Hb was compared to the manufacturer’s reference using the Student’s t-test. The data are presented as mean ± standard deviation (Table 1). Internal reference: The Advia-120 exhibited a high precision and linearity for Hb resulting in a reference Hb concentration of 12.22 ± 0.11 g/dL. Both the ABL-735 and HemoCue demonstrated a good accuracy of blood Hb compared to the reference, whereas the accuracy of the HemaVet readings were significantly lower by 9%–18% (p
