Simultaneous measurement of total hemoglobin and its derivatives in blood using CO-oximeters: analytical principles; their application in selecting analytical wavelengths and reference methods; a comparison of the results of the choices made.

Optical methods of quantifying total hemoglobin (tHb), applying the principles of the Lambert-Beer law, have been used both on untreated whole blood and on blood mixed with chemicals to form a stable chromophore, since the earliest days of laboratory medicine. The same principles may be applied for quantitation of the individual hemoglobin derivatives, such as oxyhemoglobin (O2Hb) and deoxyhemoglobin (HHb)1, as well as the non-oxygen transporting "dyshemoglobins", including carboxyhemoglobin (COHb) and methemoglobin (MetHb). The total hemoglobin measurement is typically carried out using a light source with a broad band of visible wavelengths. However, measurement of the derivatives requires using discrete, narrow bands of light in order to differentiate between the small differences of light absorbed by the individual derivatives. Either general-purpose, narrow band-pass spectrophotometers, or special-purpose photometers utilizing a set of fixed wavelengths, commonly referred to as "CO-oximeters" are suitable. Rapid, direct, photometric quantification of the derivatives, necessary in the clinical environment, relies on the specific light absorption characteristics of each hemoglobin derivative at the wavelengths selected, which in turn requires independent and exact knowledge of the concentrations of each entity in reference materials. This report examines the process involved in the selection of wavelengths and reference methods, contrasts the effects of the choices made and discusses some implications and limitations for routine measurement.