SPECTRUM ANALYSIS OF OLD BLOOD-STAINS. BY S. WATERMAN, M.D., NEWYORK. 7i'EPRINTED FROM Dr. Droicn-Seqnard9 s Archives of Scientific and Practical medicine. No. 5, 1873. NEW YORK: G. P. PUTNAM’S SONS, Twenty-third St. and Fourth Ave. 1873. SPECTRUM ANALYSIS OF OLD BLOOD-STAINS. 453 VII. SPECTRUM ANALYSIS OF OLD BLOOD-STAINS. •BY S. WATERMAN, M.D. NEW VOKK. This paper is directed to the inquiry, whether old dried blood could be spectroscopically demonstrated, and in what manner the spectrum thus obtained differed from the spectrum of blood recently dried. It will be in the recollection of the profession, that this ques- tion arose at the trial of Mr. Alley, in Boston, who stood before a jury charged with murder. The State’s medical expert stated in his testimony that old blood-spots could not be distinguished from blood- spots of recent date. I intend to prove that we can so distinguish, and that, in this respect, the statement of the expert was at variance with the exact results presented by spectral analysis. Were I to rely on authority alone, I could quote such well-known physicists as Hoppe-Seyler, Preyer, Gorup-Besonez, Kuehne and others, who, in various publications, have pointed out the spectral appearances of old blood. But I have made extended and patient investigations myself, for a number of years, and had known these varied appearances long before I became acquainted with the views of these writers. I had two specimens of old blood with which I experimented. One was four years old, and was abstracted by venesection from a female during parturition; the other was solved out from a piece of the blood- stained shirt of the murdered man Nathan, about two years old. In order to ascertain the peculiar optic relations of old blood, it should be brought to a proper state of solution. If the blood is in lumps, it ought to be finely pulverized in a clean mortar, and distilled water added, at a temperature of 6o° F. at the highest. A lower tempera- ture is preferable, but it takes longer time to solve the blood, its haemato-crystallin having lost its solubility to a very great extent by age. Ten grains of dried blood to an ounce of water gives a proper solution to experiment upon. Having intimately mixed the powdered blood and the water, set the solution aside for t2 hours, at a low tem- perature, and then filter. Let me here remind those desirous to 454 S. WATERMAN. investigate, that the more clear the solution is, the more distinct and satisfactory will be the modification of the spectrum. Repeated filtra- tion may become necessary in some cases, especially if the tempera- ture is above 6o° F. Your glass tube, or piano-parallel vessel, must be scrupulously clean also. Having made your fluid as clear as possible, submit it to the spectrum test. Note the band in red coincident and even overlapping the C line. (See Spectrum No. i.) It is never visible when fresh blood is examined, and is there- fore characteristic of old Hood. With the solution concentrated as advised above, you behold this band very well defined; all light ab- sorbed from D to H, with an extension of the red part of the spec- trum amounting to several degrees. Having observed this band, dilute your’ solution 50 per cent. The green part of the spectrum becomes visible, and the two characteristic bands of fresh blood, between D and E, appear dimly outlined, and the rest of the spectrum still overcast beyond the second band at E. Set your solution aside for another 24 hours, at the common tem- perature, and then examine again. Look at the remarkable change that has taken place. At first, when only the dark band in red was visible, it seemed as if the blood and its haemato-crystallin had lost its power to absorb oxygen from the air; it seemed as if by age a complete necrosis or death of the blood had ensued. But now, be- hold ! the two cheerful bands, characteristic of oxyhaemato-crystal- lin, have reappeared—a resurrection from death. It may need some further dilution to see them well (see Spectrum No. 2); said dila- tion will cause a corresponding diminution in the distinctness of out- line and depth of shade of the line at C, which is, as it were, waning away. The sign of death yields to the sign of life. Let your solution stand another 12 or 24 hours in a closed vessel, and on examination spectroscopically mark how the three bands have entirely disappeared, and that between D and E you now behold a dark, broad band, due to deoxidized haemato crystallin (see Spec- trum No. 3). What has caused this change ? Simply your solution has entered a putrefactive process, evolving sulphuretted hydrogen and other gases of decomposition, which in their turn have absorbed or abstracted all the oxygen of the hcemato-crystallin. You have indeed a fair specimen of reduced oxyhaemato-crystallin before you. To show that this is precisely the case, open now your glass vessel, pour out your solution, and shake it up in some larger glass vessel, with the air of the atmosphere. On submitting the solution again to the spectroscope you will find the broad band of reduced haemato- rrvstallin has disappeared, and our old dear friends, the two bands SPECTRUM ANALYSIS OF OLD BLOOD-STAINS. 455 between D and E, have reappeared (see Spectrum No. 4). They look darker and more defined than ever. Your solution, or the crys- tallizable material in it, has greedily absorbed and saturated itself with oxygen, showing that, despite long years of death apparent, the haemato-crystallin has preserved its integrity, and its capacity, when brought again in the proper conditions, to renew its vital mission. The solution at this stage will bear dilution, and will bring out the absorption-bands with rare clearness and depth of shading. You can repeat the last-mentioned process many a time, and I have not yet ascertained the limit of this absorbing power, although 1 have satisfied myself that it gradually diminishes. The next question which arises is, To what changes in the blood is this line coincident with C due ? Is it due to hsemin, which also shows a band in red between B and C ? Considering that hsemin is not a physiological ingredient of the blood, but altogether an artificial one, resulting from the action of glacial acetic acid upon blood, we must exclude the proposition that this band is due to hsemin. With regard to methsemoglobin, it is necessary to understand that no isolated crystalline body bearing that name has yet been produced. Hoppe-Seyler says : “ It is a substance intermediate between hsemato- crystallin and haematin, an intermediate product of the spontaneous transmutation of haemato-crystallin into haematin and albuminous sub- stances.” (See Handbuch der physiologisch-pathologisch-chemischen Analise, p. 220.) He places its absorption-band near C, in conjunc- tion with the two absorption-bands between D and E. Kuehne thinks that methaemoglobin is only oxyhaemato-crystallin mixed with haematin, which latter substance results from the action of butyric and formic acids, which are generated in small quantities during the decomposition of blood. Again, in his Medicinische Chemische Untersuchungen, Berlin, 1871, p. 378, Hoppe-Seyler says : “ In addition to the fatty acids I formerly believed in a substance as the spontaneous decomposition of haemato-crystallin, which I named methaemoglobin, but further in- vestigation has convinced me, that at the spontaneous decomposition haematin and albuminous substances formed at the same time.” Preyer says, die Blutcrystalle, p. 196, that methaemoglobin may be said to be a mixture of albumen, haematin and iron oxydul in very weak acidulated solution, and when thus “Viewed, the term methaemoglobin may be considered a superfluous designation. For the present he thinks we ought to retain this designation, however, because of its peculiar absorption-band, and because the solution coagulates. 456 S. WATERMAN. Under these circumstances, I am not inclined to ascribe the peculiar spectrum to methcemoglobin, because, also, in addition to the foregoing obscurity of what methcemoglobin actually is, the chemical.analysis made by me of a solution of dried blood does not correspond with the account given by Hoppe-Seyler, Preyer, Kuehne and others, nor does the position of the band, as given by these authors, correspond with the position, accurately measured by me, which covers and coincides with the line C. It is also of importance to note that when a solution of dried blood is freshly prepared, this line in C is alone visible, and that the two bands due to oxyhasmato-crystallin become only visible subsequently, when the solution has had a chance to absorb oxygen from the air, as shown by the description given above. But suppose, for argument’s sake, that there is such a substance as methaemoglobin. According to Hoppe-Seyler, it is obtained from a solution of haemato-crystallin, and not from dried blood. See also Preyer, die JSlutcrystalle, p. 191. I could not verify the results of ’-'Ppe-Seyler that the band at C could be intensified by precipitating hition with acetate of lead (Preyer says a solution of acetate uses no turbidity, nor any precipitation), filtering the pre- dion with water, and chymolysis by means of soda-car- SPECTRUM ANALYSIS OF OLD BLOOD-STAINS. 457 bonate, until all the coloring material is fully dissolved out. On the contrary, I found that after this procedure the band at C had become narrower and more nebulous, and was inferior, in depth and breadth to that of the original solution. Our next inquiry is, then, whether this band is due to haematin. Dr. A. H. Gallatin, in his very able paper before the Academy. October sth, 1871, quoted several authorities to show that this band was due to brown haematin. As haematin presents a number of spectra, we may well consider first the acid haematin spectrum which, according to Thudicum, shows four absorption-bands, one of which corresponds to our band coincident with the C line. Preyer assigns this beautiful spectrum to haematin. I have not been able to discover in the solution of old dried blood more than three bands, but this may be owing to the imperfection of my instruments, or to insufficient illuminating power, as one of the bands is very fine, like a sun-line. Lancaster and others have pointed out the fact that the position of the band in red can be influenced by the strength of the acid employed; the stronger the acid, the more considerable will be the displacement of the absorption-band towards the red end of the spectrum. Whilst this haematin band does not exactly have the position of our band peculiar to old blood, there is still another difference in the position of the two bands between D and E quite sufficient to justify us in rejecting the proposition that this spectrum of old blood is due to acid haematin. The two bands of reduced haematin occupy dif- ferent positions than those of oxyhaemato-crystallin ; they differ also in size and other peculiarities. Thus the first band of reduced hcema- tin between D and E is broader than the second, whilst the bands of oxyhaemato-crystallin are quite the reverse. So also do they dif- fer in the order of their appearance. With oxyhaemato-crystallin the two bands appear simultaneously, and, as already stated, when fully developed, the band near E is the broadest, but is not so well defined as its narrower mate. With reduced haematin the first band is already black and intense when the second begins to appear. The spectrum (or spectra) of old dried blood passes through changes very peculiar, and not in any way resembling those of haematin. If haematin is at all present in dried old blood it is difficult to un- derstand why the oxyhaemato-crystallin bands and not the haematin bands should prevail, and why the broad band of reduced haemato- crystallin should finally ensue, and not the two bands of reduced haematin, and how out of this broad band the two oxyhaemato- crystallin bands should reappear, showing the latter in its full vital- ized integrity. 458 S. WATERMAN. This band in red is probably due to the action of the various gases which are evolved during the process of drying, and which betray their evanescent character when such dried blood is solved in water and exposed to the verifying effects of oxygen. Hoppe-Seyler describes a spectrum of blood when acted upon by sulphuretted hydrogen. It gives the two normal blood-bands and a third in red near C (see Spectrum No, 5); the absorption is increased of all the blue and green part of the spectrum, so that all color is cut off beyond the third band at E (see Spectrum No. 5), It seems to me this spectrum answers best to that of old dried blood, and until we learn more of the laws and optical relations of putrefactive processes we may accept it as a provisional explanation of the spectral phenomena above described. A chemical test for old blood-stains is communicated by Gorup- Besanez, Anleitung zur Zoochemischen Analyse, on the authority of Pfafif, who recommends to use a solution of arsenious acid, i part to 120 of water. Fresh blood-spots are solved in a few minutes ; blood of one or two days old requires 15 minutes; of four or six months, 3to 4 hours; and, when nine year old, 4to 8 hours are re- quired. ARCHIVES OF Scientific and Practical Medicine. 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