IX. "On the Disappearance of the Leucocytes from the Blood, after Injection of Peptone." By Surgeon-Captain DAVID BRUCE, A.M.S., Army Medical School, Netley. Communicated by Professor HORSLEY, F.R.S. Received February 21, 1894.

As is well known, the injection of a solution of peptone into the circulation of certain animals is followed immediately by a very considerable diminution in the number of white blood corpuscles in the circulating blood. Some investigators who have written on this subject ascribe this diminution to the destruction and breaking down of the leucocytes in the blood plasma.

As this theory appeared to me to rest on the very slenderest evidence, and as it seemed to me much more natural to believe that a temporary withdrawal of the leucocytes into the internal organs took place, I was led to attempt the enumeration of the white blood corpuscles in sections of the various organs before and after the injection of peptone.

If the theory of destruction is true, then the leucocytes ought to be found in fewer numbers in the organs as well as in the blood, whereas, if the theory of temporary withdrawal be the true one, then an augmentation in their number should be seen in the sections.

For the purpose of this enumeration I used rabbits of equal weight, and proved to have a normal number of white blood corpuscles by examination of samples of blood taken from the ear.

Six rabbits were taken. The organs of two of these were examined without previous injection of peptone. The third, with a normal number of white blood corpuscles, was killed 3 hours after the injection of the peptone solution, when -ths of the white blood corpuscles had disappeared from the circulating blood.

The fourth, also having a normal number of white blood corpuscles, was killed 5 seconds after injection, when almost all the leucocytes had disappeared, at least from the blood of the heart.

In the fifth and sixth a leucocytosis was first caused by the injection of appropriate fluids and the animals killed 5 seconds after the injection of peptone.

As it seemed to me impossible to be certain of recognising all the varieties of the leucocytes in sections of the organs, I restricted myself to enumerating what I know as the polynuclear variety, since this is the variety which disappears most completely from the blood after the injection of peptone and many other substances, and which can be very readily recognised in sections after appropriate staining.

I may mention here that for practical purposes and without prejudice as to their origin, I adopt the classification of the leucocytes of the rabbit's blood into four varieties:

:

A. The Eosinophilous, constituting on an average 2 per cent. of the total leucocytes, with an irregular-shaped nucleus and large oval shaped granules, which stain readily in eosine.

B. The Polynuclear, 51 per cent., with intensely staining polymorphous nucleus, having the appearance of several nuclei united by narrow thread-like processes, the protoplasm of the cell containing fine granules which also stain in eosine.

C. The Myelocytes, 16 per cent. Difficult in every case to rigidly separate from the fourth variety, but defined as mononuclear cells, the nucleus of which stains badly, and is surrounded by comparatively a large amount of non-granular protoplasm.

D. The Lymphocytes, 31 per cent. Mononuclear cells, little larger than a red blood corpuscle, with intensely staining nucleus and narrow rim of protoplasm.

The preliminary enumeration of the leucocytes in the blood of the ear or heart was made by diluting the blood 200 times with an 8 per cent. magnesium sulphate solution, to which sufficient gentian violet had been added to stain the white blood corpuscles, and not by estimating their proportion to the red blood corpuscles, which seems to me a most unsound method. The number of white blood corpuscles in 300 squares of a Gower's hæmocytometer were then counted and this number multiplied by 333, which gives approximately the number in a cubic millimetre.

Three samples of blood are taken, two of which are counted, and, if the counts are sufficiently close, an average made. In the event of there being a marked discrepancy, the third sample is counted and an average of the two most alike taken, but, with care, it is seldom found necessary to use the third sample.

In regard to the fixing and hardening of the tissues for cutting, alcohol, Müller's solution, Foa's, and Flemming's solutions were used, but the best results were obtained by fixing for twenty-four hours in Flemming's strong solution, washing in running water for twentyfour hours, then through successive alcohols for forty-eight hours.

The tissues, after infiltration with paraffin, were cut as neatly as possible of the same thickness, and stained in various ways, the most successful results being got by staining for twenty-four hours in the Ehrlich-Biondi triple stain.

On taking the sections out of the stain I place them for a minute in a saturated solution of corrosive sublimate, as this seems in some way to prevent them becoming too much decolorised during the subsequent manipulations.

The white blood corpuscles contained in twenty fields of the micro

scope (Zeiss' 2 millimetres apochromatic, oil immersion, and 8 eye-piece) were counted in each section, several of which were mounted from each organ, and, as will be seen, with very striking uniformity of results.

I shall now proceed to describe the results obtained in each experi

ment.

No. 1. Control Experiment.-Rabbit, weight 2 kilos. The blood from an ear-vein was found to contain 6500 leucocytes per cubic millimetre-0 per cent. eosinophilous, 34 per cent. polynuclear, 20 per cent. myelocytes, 46 per cent. lymphocytes-of which 2210 were thus polynuclear. The animal was now killed by a blow on the head and the organs at once removed. Four sections of the spleen were found to contain 54, 47, 55, and 54 polynuclear leucocytes respectively (twenty fields of the microscope being counted in each specimen), the lung, 39 and 32, and the liver, 7, 6, and 7.

It will be seen from the above enumerations that a remarkable uniformity was found to exist in the distribution of the polynuclear leucocytes in different sections of the same organ. This, I may here mention, obtained throughout, not only in the control experiments, but also after the injection of peptone.

No. 2. Control Experiment.-Rabbit, 2 kilos. The blood from an ear-vein was found to contain 3833 polynuclear leucocytes in each cubic millimetre. Two sections of the spleen were counted and contained, in 20 fields, 62 and 74 respectively; the lung, 35 and 41; liver, 5 and 6.

No. 3. Control Experiment.—To show that the blood contained in the right and left ventricles does not differ markedly from the blood taken from an ear-vein in the number of leucocytes contained in the cubic millimetre the following experiment was made:

Rabbit, 2 kilos. The blood from an ear-vein was found to contain 8500 leucocytes in each cubic millimetre. After an hour the rabbit was killed by a blow on the head, and the heart ligatured and removed as soon after death as possible.

The blood of the right ventricle was found to contain 7200 leucocytes per cubic millimetre; that of the left, 7500.

The following experiment shows the number of polynuclear leucocytes found in sections of the organs of an ordinary normal rabbit after the leucocytes have been greatly diminished in the circulating blood by the injection of peptone.

No. 4. Rabbit, 2 kilos. Blood from the right ear contained 4015 polynuclear leucocytes. Three and a half hours after the injection into a vein of the left ear of 3.5 grams peptone, dissolved in 35 c.c. water, blood from the right ear only contained 1100 of the same variety.

Sections of the spleen prepared and examined in the same way as