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Step 2: Measure Plasma Hemoglobin (assumed to be Hemopure or Oxyglobin)

• Specimen preparation. A portion of a patient specimen is centrifuged and the amount of hemoglobin in the plasma is measured. The remainder of the specimen is used for the requested laboratory tests in a routine fashion.
• Instruments. Since OPK Biotech's HBOCs are polymerized hemoglobin, instruments which directly measure (do not calculate) hemoglobin in the range of 0.3 to >6.5 g/dL may be capable of measuring them. Only a few commercially available methods for measuring hemoglobin are published to support their accuracy in measuring Hemopure or Oxyglobin. Thus, the instrument manufacturer should be contacted for verification of its accuracy if your instrument is not listed below:
• Co-oximeter. This methodology permits accurate measurement of Hemopure and Oxyglobin (OPK Biotech, report on file). A co-oximeter is a spectrophotometer that monitors absorbance at multiple wavelengths to determine total hemoglobin concentrations.
• Hemoglobinometer. One hemoglobinometer, HemoCue-B-Haemoglobin Analyzer (Hemocue Ltd, Sheffield, UK) has been found to be accurate for measuring either Hemopure or Oxyglobin. This instrument has also been validated for use in multiple species.
• Callan MB, Giger U, Oakley DA, Scotti MV, Shofer FS. Evaluation of an automated system for hemoglobin measurement in animals. Am J Vet Res. 1992;53(10):1760-1764.
• Jahr JS, Lurie F, Driessen B, Davis JA, Gosselin R, Gunther RA. The HemoCue, a point of care B-hemoglobin photometer, measures hemoglobin concentrations accurately when mixed in vitro with canine plasma and three hemoglobin-based oxygen carriers (HBOC). Can J Anaesth. 2002 Mar;49(3):243-8.
• Lurie F, Jahr JS, Driessen B. 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 (Hemolink). Anesth Analg. 2002 Oct;95(4):870-3.
• Hematology instruments. The methodologies of hematology instruments are variable and the instrument manufacturer should be contacted. At least one hematology instrument, the Bayer Advia 120, has been shown to be accurate in measuring Hemopure (and thus also accurate for Oxyglobin):
• Kunicka J, Malin M, Zelmanovic D, Katzenberg M, Canfield W, Shapiro P, Mohandas N. Automated quantitation of hemoglobin-based blood substitutes in whole blood samples. Am J Clin Pathol. 2001 Dec;116(6):913-9.
• Hemolytic Index: A linear correlation exists between the Hemolytic Index and increasing HBOC concentrations. If a laboratory chooses to use a Hemolytic Index correlation, a laboratory-created regression equation should be used to determine the specific correction factors between the two indices. The Hitachi Modular P800 was found to correlate Hemolytic Index with Hemopure concentration:
• Moon-Massat PF, Tierney JP, Hock KG, Scott MG. Hitachi Hemolytic Index correlates with HBOC-201 concentrations: Impact on suppression of analyte results. Clin Biochem. 2007 Dec 23; [Epub ahead of print].
• Results. The measured plasma hemoglobin concentration will determine if the specimen contains more or less HBOC than the accepted maximal concentration, based on the Assay Limitation Sheet, which will allow accurate results.
• Go to Step 3.