The bright colour of haemoglobin has, from the very beginning, played a significant role in both the investigation of this compound as well as in the study of blood oxygen transport. Numerous optical methods have been developed for measuring haemoglobin concentration, oxygen saturation, and the principal dyshaemoglobins in vitro as well as in vivo. Modern applications include pulse oximeters, fibre optic oximeters, multiwavelength haemoglobin photometers ('co-oximeters') and instruments for near infrared spectroscopy in vivo. Knowledge of the light absorption spectra of the common haemoglobin derivatives is a prerequisite for the development and understanding of these techniques. In the 1960s a reference method based on the absorptivity of a single derivative (haemiglobincyanide; HiCN) at a single wavelength (540 nm) was established for measuring the total haemoglobin concentration. Thus an anchor value was provided on which the absorptivity spectra of all other haemoglobin derivatives could be based. This monograph presents absorption spectra and absorptivity data in the wavelength range of 480 to 1000 nm of the major haemoglobin derivatives for human adult and foetal haemoglobin and for haemoglobin of several animals (cow, dog, horse, pig, rat, and adult and foetal sheep). A detailed description of the methods used to acquire these data has been included to allow future investigators to reproduce and expand on the data. The second part of the monograph includes chapters on the principles and development, in historical perspective, of the principal methods for measuring total haemoglobin concentration for two, three, and multi-component analysis of haemoglobin derivatives, and for blood oxygen saturation measurement. Accurate quantitative data pertaining to haemoglobin in human blood are presented, together with a description of methods for measuring haemoglobin oxygen capacity and oxygen affinity. These chapters have been written with a view to foster knowledge and insight concerning the principles, purposes, and limitations of the various methods of all who use these methods in research and patient care.
Foreword Preface INTRODUCTION Visual spectrophotometry Photoelectric spectrophotometry Diode array spectrophotometry Biomedical applications DEFINITIONS AND TERMINOLOGY Spectrophotometry Haemoglobin and haemoglobin derivatives Oxygen carrying properties of mammalian blood SPECTROPHOTOMETRY The laws of absorption of radiant energy Applications of Lambert-Beer's law Instrumentation Spectrophotometer performance TOTAL HAEMOGLOBIN CONCENTRATION ABSORPTIVITY AT 540 NM OF HAEMIGLOBINCYANIDE Preparation of a haemoglobin stock solution Determination of the iron concentration of the haemoglobin stock solution Determination of the absorbance at 540 nm of the haemoglobin stock solution after conversion to HiCN Calculation of the absorptivity at 540 nm of HiCN HiCN(540) as found for human and animal haemoglobin PREPARATION OF HAEMOGLOBIN DERIVATIVES Specimen procurement and handling Tonometry, erythrolysis and filtration Preparation of Hhb, O2Hb, and COHb Preparation of Hi, HiCN, and Shb DETERMINATION OF ABSORPTION SPECTRA Lightpath length Absorbance measurement Measurement of total haemoglobin Measurement of foetal haemoglobin Absorptivity calculation Influence of temperature and pH ABSORPTION SCPECTRA OF HUMAN HBA AND HBF Absorptivity of HbA: standard procedure Absorptivity of HbA: SFH procedure Absorptivity of HbF: standard procedure Comments Spectral characteristics of modified human haemoglobin ABSORPTION SPECTRA OF DOG HAEMOGLOBIN ABSORPTION SPECTRA OF RAT HAEMOGLOBIN ABSORPTION SPECTRA OF BOVINE HAEMOGLOBIN ABSORPTION SPECTRA OF PIG HAEMOGLOBIN ABSORPTION SPECTRA OF HORSE HAEMOGLOBIN ABSORPTION SPECTRA OF SHEEP HAEMOGLOBIN Absorptivity of adult haemoglobin Absorptivity of foetal haemoglobin Comments COMMENTS ON THE DETERMINATION OF ABSORPTION SPECTRA OF HAEMOGLOBIN HAEMOGLOBINOMETRY A short history The reference point of standardised haemoglobinometry The haemiglobincyanide method Haemiglobincyanide standards Routine haemoglobinometry MULTICOMPONENT ANALYSIS OF HAEMOGLOBIN DERIVATIVES The lengthy road to MCA Selected two-wavelength methods for O2Hb and COHb Multicomponent analysis Multiwavelength haemoglobin photometers MCA including Hi and HiCN Current developments OXIMETRY AND RELATED TECHNIQUES Development of oximetry Cuvette oximetry and denitometry Syllectometry Fibre optic oximetry Pulse oximetry Near infrared specroscopy THE OXYGEN BINDING CAPACITY OF HUMAN HAEMOGLOBIN Early determinations of the oxygen binding capacity of human haemoglobin Reference method for total oxygen in blood Redetermination of the oxygen binding capacity of human haemoglobin Routine determination of the oxygen capacity of human blood THE OXYGEN AFFINITY OF HUMAN HAEMOGLOBIN Instruments and procedures The normal oxygen dissociation curve of human blood Effect of temperature The Bohr and Haldane effects Effect of 2,3-diphosphoglycerate Effect of dyshaemoglobins Conclusions References Abbreviations and symbols Index