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Artif Cells Nanomed Biotechnol. 2017 Feb;45(1):58-62. Epub 2016 Mar 2.

Determination of methemoglobin and hemoglobin levels in small volume samples.

Author information

1
a NeuroTrauma Department, Operational and Undersea Medicine Directorate, Naval Medical Research Center , Silver Spring, MD , USA.
2
b Department of Surgery , Uniformed Services University of the Health Sciences , Bethesda , MD , USA.

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.

KEYWORDS:

96-well-plate; Drabkin; Evelyn-Malloy; HBOC; cyanide

PMID:
26934005
DOI:
10.3109/21691401.2016.1138490
[Indexed for MEDLINE]

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