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white to every 500 or 600 red corpuscles. There are certain diseases which change very materially this proportion of the different forms of leukocytes, and even in health there is a relevant increase a “leukocytosis” immediately after meals and after cold baths.
Cabot enumerates all of the different physiological leukocytoses as follows: (1) leukocytosis of the new-born; (2) leukocytosis of digestion; (3) leukocytosis of pregnancy (4) leukocytosis of postpartum; (5) leukocytosis after violent exercise, massage and cold baths; and (6) leukocytosis of the moribund state.
In certain diseases we find that there is a more or less increase of the white-blood corpuscles, or in other words a leukocytosis, such as in the different form of leukemia, scarlet fever, diphtheria, pneumonia, cholera, erysipelas, smallpox, glanders, etc., while in other kinds of diseases there is a reduction of the leukocytes (leukopenia), as in malarial fever, measles, consumption, etc.
Believing that an increase in the number of leukocytes might be of advantage in the treatment of consumption, Vaughan and others for a time used nuclein, a product of the nuclei of cells, especially of the polynuclear leukocytes, which seemed to produce a slight leukocytosis which it was believed occurred principally amongst the polymorphonuclear variety. Vaughan and others believed that the germicidal constituent of blood serum was due to nucleins. 3
MOVEMENT OF LEUKOCYTES.
If we withdraw a drop of fresh blood and place it under the microscope, we will find that some, but not all of the white-blood corpuscles, by means of pseudopodia which they project outwards from their body move themselves from place to place, constantly pulling the nuclei in different positions with reference to the body of the leukocytes.
The writer of this paper at one time saw one of these leukocytes by means of its pseudopodia surround or envelop a malarial parasite contained in a red-blood corpuscle, and can say that it is a beautiful picture to watch this miniature warfare going on.
The greatest activity of the amoeboid movement occurs amongst the polynuclear variety, and these are the most destructive to bacteria. In other words, the polynuclear variety are the soldiers on guard against the invasion of disease-producing bacteria.
THE OPSONIC INDEX.
In 1895, Denys and Lecleft called attention to the effect of blood serum on phagocytosis. They found that by certain experi
ments they could alter microbes in such a way that they could be subsequently ingested by the leukocytes. They found that an ani-' mal which had been immunized fights bacteria first by the direct action of its serum; and second by its leukocytes. In this action they found that the serum was the most powerful, and that the activity of the leukocytes in destroying bacteria depended upon the activity of the blood serum. This active principle of the blood serum which cripples the disease-producing bacteria so that they may be subsequently devoured by the leukocytes, has now become known as "opsonin.” Besides this action the opsonins stimulate phagocytosis. The number of bacteria which may be devoured by each individual polynuclear leukocyte, it is held, depends upon the amount of opsonin contained in the blood serum. So that in order to get rid of a disease dependent upon any certain kind of microorganism, it is held, we should strive to increase the opsonin, and leave the crippled microbes to the mercy of the phagocyting leukocytes.
The power exerted by the polymorphonuclear leukocytes in engulfing the disease micro-organisms (the number that may be taken up per leukocyte), as it occurs in a given individual during health, is compared to the number of micro-organisms per polymorphonuclear leukocyte during disease furnishes what is now termed the “opsonic index.”
In 1903, Wright and Douglas introduced the word "opsonin” (opsono, I prepare the food for) to characterize the substance in normal serum which they believed prepared the microbes for ingestion by the phagocytes. They were able to show that this substance exists in the serum, that it is thermolabile, being destroyed at 60° C. in ten minutes, and that it acts on the bacteria and not on the leukocytes.
In 1904, Neufeld and Rimpau in an article spoke of the two elements in immune sera (i. e., antitoxin and bactericidal substances), and claimed that they had found a third substance which sensitized bacteria but did not act on leukocytes. Neufeld and Rimpau treated leukocytes with antistreptococcus serum, for a certain time suspended them in normal serum and found that they did not ingest virulent streptococci. On the other hand, after treating virulent streptococci with antistreptococcus serum and then washing them free of salt solution, they found that the whiteblood corpuscles could then actively ingest these sensitized microbes. They obtained corresponding results with pneumococci. They called their sensitizing substances "bacterio-trophic” sub
stances, but Wright and Douglas apply the name of "immune op'sonins."
METHOD OF DETERMINING THE OPSONIN INDEX.
Several methods are now employed to determine the opsonic index, or the opsonic content of the blood serum.
In 1902, Leishman devised a method for quantitatively estimating phagocytosis. Later, Wright modified Leishman's method, as follows:
Wright took equal volumes of (1) a bacterial emulsion in salt solution; (2) washed blood corpuscles; and (3) the serum to be tested, all of which are contained in a capillary pipette, mixed thoroughly and placed in an incubator for fifteen or twenty minutes at 37° C. A drop of this bacterial emulsion is then blown out on a slide and a smear fixed and stained. The enumeration of the bacteria in 20 to 50 polymorphonuclear neutrophiles is made under an oil immersion lens and an average number of contained bacteria per leukocyte obtained. The ratio of this average to that for a normal serum, using the same emulsion of leukocytes and corpuscles constitute the “opsonic index.”
Working after this manner, Wright has obtained the normal opsonic index for staphylococci, streptococci, pneumococci, gonococci, colon bacillus, tubercle bacillus, and other varieties of bacteria.
Bowditch and his coworkers (Ditman and Bradley), working with staphylococci after placing an emulsion of washed corpuscles and blood serum in an incubator, removed portions of it at different intervals, and obtained the following results:
After five minutes there was an average of 5.7 staphylococci per leukocyte.
After ten minutes there was an average of 8.2 staphylococci per leukocyte.
After twenty minutes there was an average of 14.5 staphylococci per leukocyte.
After one hour there was an average of 28.5 staphylococci per leukocyte.
After two hours the contained bacteria in each leukocyte were too numerous to be counted.
These writers state that with practice two persons can count an average of about five slides an hour. To make an index of two or three sera to a single germ should not take more than two hours.
Simon' believes that certain errors might result in obtaining the opsonic index outlined by Wright, and is under the impression that more accurate results can be obtained by estimating the percentage of phagocyting leukocytes. By comparing the figure thus obtained with the figure corresponding to a specimen of pooled normal blood serum, terming the latter 1, an index is obtained which is directly comparable to Wright's method.
Ambergs describes the method of Simon and Lamar as follows: "The serum is diluted with a 1 per cent saline solution in a proportion of 1 to 20 by means of a pipette used in the counting of white-blood corpuscles. Then twice nine divisions of the pipette are put in a small glass tube. In preparing a dilution of 1 to 40, nine divisions of the dilution 1 to 20 are mixed with nine divisions of the saline solution. The tubes are then charged with bacteria directly from the agar tube. Lastly, equivalent of six divisions of an emulsion of blood corpuscles containing the leukocytes is added from a specially calibrated pipette. In this manner the total amount of fluid contained in each tube corresponds to twenty-four divisions of the pipette. The blood corpuscle emulsion is obtained in the usual way. The blood is taken up in a 0.1 per cent solution of ammonium oxalate in 1 per cent sodium chloride solution to prevent coagulation. The blood corpuscles are sedimented and washed three times with a 1 per cent saline solution with the help of the centrifuge. Finally, the supernatant fluid is pitted off as completely as possible and the sediment is stirred up. The charged tubes are kept in the incubator at a temperature of 37° C. for one-half hour. Smears are made and then stained with aqueous alkaline methylene blue, so that the red corpuscles remain unstained. Then the percentage of leukocytes which have taken up bacteria is determined. In most cases fifty leukocytes should be counted.
EFFECT OF CERTAIN CHEMICALS UPON THE OPSONIC INDEX.
Bowditch and his coworkers state that chemically opsonins may be neutralized or bound by various salt solutions, such as Ca, Ba, MgCl,, K,SO,, NaHCO3, lactic acid, and chloroform, and so prevented from acting on bacteria.
There are certain other conditions which modify opsonins, such as age, sunlight, heat, dilution, and by the drugs above mentioned.
The administration of potassium iodid produces no effect upon the opsonic index.
Huggard and Morland, in 1905, after injecting nuclein, found that the opsonic index dropped for two days and then rose above normal. These observers found that the lowest opsonic index existed in cases of tuberculosis who were heavy smokers.
RELATION OF OPSONINS TO NUMBER OF LEUKOCYTES.
Bowditch and his coworkers, quoting from other writers, further state that as leukocytosis was almost universal in the course of infections it might be thought that some relationship might exist between the quantity and quality of the leukocytes and the opsonic content of the serum. This supposition, however, is not a fact, and the experiments of Huggard, Morland, Bulloch and Ledingham all.go to prove that there is no direct relationship existing between the opsonic index and the quantity and quality of the leukocytosis.
Experiments more recently made show that the opsonins, whatever their real nature may be, exist in the blood serum, and not in the leukocytes. Opsonins are not a product of leukocytes.
The experiments of Denys and Leclef, Hektoen, Ruediger and Horton show, however, that the increase of virulence of a certain organism goes hand in hand with its resistance to phagocytosis. That is to say, when phagocytic action is below normal, the disease is more virulent.
According to this view, to remedy such a condition and to get rid of a disease, it should be the endeavor of the physician to increase or restore this phagocytic action of the leukocytes up to the normal in combating that special microbe which causes the dis
At the present time we know that there are different kinds of bacterial antitropins, such as antitoxins, agglutinins, precipitins, lysins and opsonins.
THE OPSONIC INDEX IN HEALTH AND DISEASE.
Bulloch stated that the tuberculo-opsonic index in sixty-six healthy individuals was 0.95, with a maximum of 1.20 and a minimum of 0.80. Bowditch and others give the average normal opsonic index of 0.98, with a maximum of 1.25 and minimum of 0.73. Investigators are now at work in various parts of the world endeavoring to establish the opsonic index in many diseases, such as croupous pneumonia, staphylococcic and streptococcic infection, gonorrheal rheumatism, typhoid fever, syphilis, cancer, pleurisy, chronic cystitis, acne, gout and many other diseases. These investigations may have a direct influence regarding our prognosis in these diseases.
Since these investigations more prominently brought forth by Wright, physicians have begun to report good results in the treat