This chapter is vaguely related to Section F8(vii) from the 2017 CICM Primary Syllabus, which expects the exam candidates to be able to "describe physiology and consequences of abnormal haemoglobin". The act of stretching this syllabus item to the discussion of haemoglobin spectrophotometry is justified on the premise that one should understand how one is measuring one's blood results. The general principles of using absorption spectrophotometry to identify and measure the concentrations of haemoglobin species are discussed elsewhere. This chapter focuses on the use of these principles to measure the concentration of methaemoglobin, expressed by the ABG machine as FMetHb.

Measurement of methaemoglobin using absorption spectrophotometry

Methaemoglobin, with its useless Fe3+ atoms, presents a starkly different absorption spectrum when compared to oxyhaemoglobin, the dominant species (going by concentration)

comparion of oxyhaemoglobin and methaemoglobin absorption spectra

Thus, in a situation when it is hopelessly outnumbered by oxyhaemoglobin molecules, methaemoglobin makes its presence felt by absorbing strongly in the 600-650 nm range of wavelengths, bordering on infrared.

This provides us with a predictable change to look for in the graph of the ε/λ relationship for total haemoglobin absorption. This predictable change can be demonstrated in empirical measurements. For instance, the graphs below were first published in "Advances in Clinical Chemistry"; Volume 8- specifically, the chapter by E.J. van Kampen and W.G. Zijlstra, "Determination of haemoglobin and its derivatives". The authors measured a series of dilute blood samples with different FMetHb concentrations:

influence of methaemoglobin on the absorption spectrum of a blood sample

Not only does the upper ranged of wavelength differ predictably according to the concentration of methaemoglobin, but the laboratory data is supported beautifully by the findings from a real clinical scenario. The authors quote a case report:

"A 40-year-old fruit sprayer was brought to the hospital one evening with severe cyanosis. During the day he had sprayed an orchard without a mask; using nitrobenzene. The absorption spectrum of a blood sample recorded on admission is shown in Fig. 17. From this spectrum the Hi concentration was estimated to be near 20%. A prompt improvement followed intravenous injection of 1 g sodium ascorbate and 1 g methylene blue. "

No reference is provided, but the case is probably analogous to this one. This just goes to show that nitrobenzene (previously known as Oil of Mirbane) had good reasons for being banned almost everywhere, and why it features high on my list of substances I would never want to get a facefull of.

Spurious methaemoglobinaemia

The ABG spectrophotometer can occasionally become confused. There are several situations in which tit may offer a spurious reading; i.e. confabulate a high methaemoglobin level were there is in fact none. There are two common

  • Hyperlipidaemia which in general tends to produce unusual reading in the spectrophotometer, giving rise to abnormally high total haemoglobin readings and negative carboxyhaemoglobin numbers. These extra fatty acids tend to scatter the light, interfering with the absorption spectrum and giving the impression that a lot more light has been absorbed.
  • Isosulfant blue, otherwise known as "patent blue" - a dye used in surgery to identify lymphatic vessels, eg. for sentinel node biopsy

References

Device-specific information in all these ABG pages refers to the ABG machine used in my home unit.

Other machines may have different reference ranges and different symbols.

For my ABG analyser, one can examine this handy operations manual.

There is also an even more handy reference manual, but one needs to be an owner of this equipment before one can get hold of it. Its called the "989-963I ABL800 Reference Manual"

Nastuk, William L., ed. Special Methods: Physical Techniques in Biological Research. Elsevier, 1962.

von Kompen, E. J. "Spectrophotometry of hemoglobin and hemoglobin derivatives." Advances in clinical chemistry 23 (1983): 199.

Zwart, A. "Spectrophotometry of hemoglobin: various perspectives." Clinical chemistry 39.8 (1993): 1570-1572.

H Sobotka et al, "Advances in Clinical Chemistry"; Volume 8- specifically, the chapter by E.J. van Kampen and W.G. Zijlstra, "Determination of haemoglobin and its derivatives"

- The graphs from this chapter were reproduced above without any permission from the authors, but in the spirit of respect and admiration for their work.

Stevenson, A., and Roy P. Forbes. "Nitrobenzene poisoning: Report of a case due to exterminator spray." The Journal of Pediatrics 21.2 (1942): 224-228.

Hogarth, C. W. "A case of poisoning by oil of mirbane (nitro-benzol)." British medical journal 1.2665 (1912): 183.

Spurzem, J. R., H. W. Bonekat, and J. W. Shigeoka. "Factitious methemoglobinemia caused by hyperlipemia." CHEST Journal 86.1 (1984): 84-86.

Burgoyne, Laura L., et al. "Isosulfan blue causes factitious methemoglobinemia in an infant" Pediatric Anesthesia 15.12 (2005): 1116-1119.

Straub, K. D., and W. S. Lynn. "Turbidity effects in dual wavelength spectrophometry." Biochimica et Biophysica Acta (BBA)-Biophysics including Photosynthesis 94.1 (1965): 304-306.