Text-book of forensic medicine and toxicology

by R. J. M. Buchanan

The spectra of hæmoglobin and its derivatives are characteristic, and afford conclusive evidence of the presence of blood. The spectra must be recognised, however, in more than one condition. Other substances may yield spectra very similar to that of oxyhæmoglobin, but when subjected to certain tests they do not alter in the same way. They cannot be made to give the spectra of reduced hæmoglobin and reduced hæmatin, and any colouring matter which may be made to yield the spectra of reduced hæmoglobin and reduced hæmatin is derived from blood.

Blood Spectra

1. Oxidised hæmoglobin (O₂Hb) is characterised by the presence in its solar spectrum of two absorption bands between the D and E lines. The first band commences at the D line and extends a short distance towards the E. The second commences at a little distance from it, and terminates at the E line; it is about twice the breadth of the first. The band at D is more defined than the other ([Fig. 15, 1]).

2. Deoxidised or reduced hæmoglobin presents one broad band occupying almost the whole of the space between D and E slightly to the left of these lines ([Fig. 15, 2]).

3. Methæmoglobin presents two bands between D and E, in the same position as those of O₂Hb, but in addition a third band between C and D and near to the former ([Fig. 15, 3]).

A solution of oxyhæmoglobin or methæmoglobin may be reduced by the addition of a reducing agent, such as Stokes‘ reagent, consisting of ferrous sulphate with a small quantity of tartaric acid dissolved in water and rendered alkaline at the time of using with ammonia, or, better still, by the addition of ammonium sulphide. The spectrum will change to that of reduced hæmoglobin.

4. Acid hæmatin presents a spectrum with a band between D and E, commencing at a little distance from D and ending at E, also a narrower band between C and D and commencing at C. It is a difficult spectrum to obtain.

5. Alkaline hæmatin presents a spectrum with a single band between C and D near to the D line. It is more difficult to obtain than the spectrum of acid hæmatin.

It is not necessary, however, to obtain these spectra, viz. 5 and 6, but it is necessary to reduce solutions of either acid or alkaline hæmatin in order to obtain the spectrum of reduced hæmatin. To do so proceed as follows. To some of the solution of colouring matter obtained from the stain add a small quantity of a 20 per cent. solution of sodium hydrate; the solution will alter in colour, and the spectrum of O₂Hb or MetHb will disappear. On adding to this solution of alkaline hæmatin a few drops of ammonium sulphide, or Stokes‘ fluid, it becomes claret-coloured, and on examination with the spectroscope the spectrum of reduced hæmatin will be seen. This is the most pronounced of all blood spectra. Its production can be hastened by gently warming the solution.

If the stain be old and already changed into hæmatin, its solution will yield the spectrum of acid hæmatin, and will give the spectrum of reduced hæmatin on the addition of ammonium sulphide or Stokes‘ fluid without previous alkalisation.