Determination of methemoglobin and hemoglobin levels in small volume samples

Francoise Arnaud*, Adam Higgins, Richard McCarron, Paula F. Moon-Massat

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

9 Scopus citations


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.

Original languageEnglish
Pages (from-to)58-62
Number of pages5
JournalArtificial Cells, Nanomedicine and Biotechnology
Issue number1
StatePublished - 2 Jan 2017
Externally publishedYes


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


Dive into the research topics of 'Determination of methemoglobin and hemoglobin levels in small volume samples'. Together they form a unique fingerprint.

Cite this