title: Determination of methemoglobin and hemoglobin levels in small volume samples.
abstract: Background/aims Hemoglobin-based oxygen carriers (HBOCs) have been previously studied as resuscitation fluids. Due to HBOCs specific molecular conformation, hemoglobin (Hb) and methemoglobin (MetHb) determination is not always possible with automated apparatus. A practical technique was designed that allows simultaneous reading of MetHb and Hb in small volume samples. Methods A spectrophotometric method for measuring low levels of MetHb and Hb from limited volume samples was developed using a 96-well-plate by downsizing the Evelyn-Malloy and Drabkin methods. Either blood or buffer samples were spiked with one of five HBOCs (HBOC-201, M101, MP4CO-NP, Sanguinate and Oxyvita C). After treatment with cyanides, the samples were read at 540, 630, and 680 nm, and Hb and MetHb results were compared to certificate-of-analysis. Results Hb levels ranging from 0.2 to 2.8 g/dl were detected accurately with the 96-well-plate method with HBOC-201. Similarly, this method accurately measured Hb from either plasma or buffer samples containing any of the HBOCs. The MetHb plasma samples with HBOC-201 were also in agreement with ABL results (R = 0.99719). MetHb from all HBOCs in buffer measured with this method was comparable to reference but the accuracy was compromised for HBOCs in blood. Conclusions A useful 96-well-plate method of measuring HBOCs' Hb was designed for small-volume plasma samples. It was accurate for measuring MetHb from samples, that contained M101, MP4CO-NP, Sanguinate, and Oxyvita C diluted in buffer. This well-plate method allows reading of batch samples, multiple replicates, and using small volumes to accommodate limited animal blood collection which would not be otherwise detected by automated instrumentation.
authors.affiliation: a NeuroTrauma Department, Operational and Undersea Medicine Directorate, Naval Medical Research Center , Silver Spring, MD , USA.|b Department of Surgery , Uniformed Services University of the Health Sciences , Bethesda , MD , USA.
authors.forename: Francoise
authors.initials: F
authors.lastname: Arnaud
authors.affiliation: a NeuroTrauma Department, Operational and Undersea Medicine Directorate, Naval Medical Research Center , Silver Spring, MD , USA.
authors.forename: Adam
authors.initials: A
authors.lastname: Higgins
authors.affiliation: a NeuroTrauma Department, Operational and Undersea Medicine Directorate, Naval Medical Research Center , Silver Spring, MD , USA.|b Department of Surgery , Uniformed Services University of the Health Sciences , Bethesda , MD , USA.
authors.forename: Richard
authors.initials: R
authors.lastname: McCarron
authors.affiliation: a NeuroTrauma Department, Operational and Undersea Medicine Directorate, Naval Medical Research Center , Silver Spring, MD , USA.
authors.forename: Paula F
authors.initials: PF
authors.lastname: Moon-Massat
mesh_terms: D000818:Animals; D008706:Methemoglobin / Q000032:analysis*; D051381:Rats
chemical_list: D008706:Methemoglobin
keywords: 96-well-plate; Drabkin; Evelyn-Malloy; HBOC; cyanide