Part 6

THE MORPHOLOGY OF THE BLOOD.

A. METHODS OF INVESTIGATION.

A glance at the history of the microscopy of the blood shews that it falls into two periods. In the first, which is especially distinguished by the work of Virchow and Max Schultze, a quantity of positive knowledge was quickly won, and the different forms of anæmia were recognised. But close upon this followed a standstill, which lasted for some decades, the cause of which lay in the circumstance that observers confined themselves to the examination of fresh blood. What in fact was to be seen with the aid of this simple method, these distinguished observers had quickly exhausted. That these methods were inadequate is best shewn by the history of leucocytosis, which after the precedent of Virchow was in general referred to an increased production on the part of the lymphatic glands; and further by the imperfect distinction between leucocytosis and incipient leukæmia, which was drawn almost exclusively from purely numerical estimations. It was only after Ehrlich had introduced the new methods of investigation by means of stained dry preparations, that the histology of the blood received the impulse for its second period.

We owe to them the exact distinction between the several kinds of white blood corpuscles, a rational definition of leukæmia, polynuclear leucocytosis, and the knowledge of the appearances of degeneration and regeneration of the red blood corpuscles, and of their degeneration in hæmoglobinæmic conditions. The same process, then, has gone on in the microscopy of the blood that we see in other branches of normal and pathological histology: by advances in method, advances in knowledge full of importance result. It is therefore little comprehensible, that an author quite recently should recommend a reversion to the old methods, and emphatically announce that he has managed to make a diagnosis in all cases, with the examination of fresh blood. At the present time, after the most important points have been cleared up by new methods, in the large majority of cases, this is not an astonishing achievement. For any difficult case (for instance the early recognition of malignant lymphoma, certain rare forms of anæmia, etc.) as the experienced know, the dry stained preparation is indispensable. The object of examining the blood, is certainly not to make a rapid diagnosis, but to investigate exactly the individual details of the blood picture. To-day, we can only take the standpoint, that everything that is to be seen in fresh specimens--apart from the quite unimportant rouleaux formation, and the amoeboid movements--can be seen equally well, and indeed much better in a stained preparation; and that there are several important details which are only made visible in the latter, and never in wet preparations.

As regards the purely technical side of the question, the examination of stained dry specimens is far more convenient than that of fresh. For it leaves us quite independent of time and place, we can keep the dried blood with few precautions for months at a time, before proceeding to further microscopic treatment; and the examination of the preparation may last as long as required, and can be repeated at any time. On the contrary, the examination of the wet preparation is only possible at the bedside, and must be conducted within so short a time, on account of the changeability of the blood, clotting, destruction of white corpuscles and so forth, that a searching investigation cannot be undertaken. In addition the preparation and staining of dried blood specimens is amongst the simplest and most convenient of the methods of clinical histology. In the interest of its wider dissemination, it will be justifiable to describe it more in detail.

We must also mention here the use of the dry preparation in the estimation of the important relation between the number of the red and of the white corpuscles; and also of the relative numbers of different kinds of white blood corpuscles.

For this purpose, faultless specimens, specially regularly spread, are indispensable. Quadratic ocular diaphragms (Ehrlich-Zeiss) are requisite, which form a series, so that the sides of the squares are as 1:2:3 ... :10, the fields therefore as 1:4:9 ... :100. The eye-piece made by Leitz after Ehrlich's directions is more convenient, in which, by a handy device, definite square fractions of the field can be obtained. The enumeration is made as follows. The white blood corpuscles are first counted in any desired field with the diaphragm no. 10, that is with the area of 100. Without changing the field, the diaphragm 1, which only leaves free a hundredth part of this area, is now put in, and the red corpuscles are counted. The field is then changed at random, and the red corpuscles counted in a portion of the area which represents the hundredth of that of the white. About 100 such counts are to be made in a specimen. The average of the red corpuscles is then multiplied by 100 and so placed in proportion with the sum of the white. If the white corpuscles are very numerous, so that counting each one in a large field is inconvenient, smaller sections of the eye-piece 81, 64, 49, etc. may be taken.

The important estimation of the percentage relation of the various forms of leucocytes is effected by the simple "typing" of several hundred cells, a count which for the practised observer is completed in less than a quarter of an hour.

To obtain unexceptionable preparations cover-slips of particular quality are necessary. They should not be thicker than 0.08 to 0.10 mm., the glass must not be brittle or faulty, and must in this thickness easily allow of considerable bending, without breaking. Every unevenness of the slip renders it useless for our purposes. The glasses must previously be particularly carefully cleaned, and all fat removed. It is generally sufficient to allow the slips to remain in ether for about half-an-hour, not covering one another. Each one still wet with ether is then wiped with soft, not coarse, linen rag or with tissue-paper. The slips now are put into alcohol for a few minutes, are dried in the same manner as from the ether, and are kept ready for use in a dust-tight watch-glass. Bearing in mind, that these cover-slips are not cut out from a flat piece but from the surface of a sphere, it is evident that only with glasses thus prepared, can it be expected that a capillary space should be formed between two of them, in which the blood spreads easily. For with the smallest unevenness or brittleness of the glass it is an impossibility for the one to fit every bend of the other. And it is only then that the slips can be drawn away one from another, without using a force which breaks them.

To avoid fresh soiling of the cover-slips, and above all the contact of the blood with the moisture coming from the finger, the cover-glass is held with forceps to receive the blood. We recommend for the under cover-glass a clamp forceps a, with broad, smooth blades; the ends may be covered with leather or blotting-paper for a distance of about 1/2 in. For the other cover-slip a very light spring forceps b, with smooth blades, sharp at the tips, is used, with which a cover-glass can be easily picked up from a flat surface. The lower slip is now fixed by one edge in the clamp forceps, and held ready in the left hand. The right hand applies the upper glass with the forceps b to the drop of blood as it exudes from the puncture, and takes it up, without touching the finger itself. The forceps b is then quickly brought to a and the slip with the little drop of blood allowed to fall lightly on the other. In glasses of the right quality the drop distributes itself spontaneously in a completely regular capillary layer. With two fingers of the right hand on the edge of the upper glass, it is now carefully pulled from the lower, which remains fixed in the clamp, without pressing or lifting. Frequently only one, the lower, shews a regular layer, but occasionally both are available for examination. During the desiccation in the air, generally complete in 10-30 seconds, the preparations must naturally be protected from any dampness (for example the breath of the patient).

The extent of surface which is covered depends on the size of the drop, the smaller the latter, the smaller the surface over which it has to be spread. Large drops are quite useless, for with them, the one cover-glass swims on the other, instead of adhering to it.

Although a written description of these manipulations makes the method seem rather intricate, yet but little practice is required to obtain an easy and sure mastery over it. We have felt compelled to describe the method minutely, since preparations so often come under our notice which, although made by scientific men, who pursue hæmatological investigations, are only to be described as technically completely inadequate.

The specimens so obtained, after they are completely dried in the air, should be kept between layers of filter-paper in well closed vessels till further treatment. In important cases, preparations of which it is desirable to keep for some considerable time, some of the specimens should be kept from atmospheric influences by covering them with a layer of paraffin. The paraffin must be removed by toluol before proceeding further. The preparations must naturally be kept in the dark.

. Fixation of the dry specimen.

All methods of staining available for the blood require the fixing of the proteids of the blood. A general formula cannot be given, since the intensity of the fixation must be regulated in accordance with the kind of stain that is chosen. Relatively slight degrees of hardening suffice for staining in simple watery solutions, for example, in the triacid fluid, and can be attained by a short, and not too intense action of several reagents. For other methods, in which solutions that are strongly acid or alkaline are employed, it is however necessary to fix the structure much more strongly. But here, too, an excess as well as an insufficiency must be guarded against. It is easy with the few staining fluids that are in use to ascertain the optimum for each.

The following means of fixation are employed.

1. Dry Heat.

A simple plate of copper on a stand is used, under one end of which burns a Bunsen flame. After some time a certain constancy in the temperature of the plate is reached, the part nearest to the flame is hottest, that farther away is cooler. By dropping water, toluol, xylol, etc. on to it, one can fairly easily ascertain that point of the plate which has reached the boiling temperature of the particular fluid.

Far more convenient is Victor Meyer's apparatus, used by chemists. This consists of a copper boiler, modified for our purpose, with a roof of thin copper-plate, perforated for the opening of the vapour tube. Small quantities of toluol are allowed to boil for a few minutes in the boiler, and the copper-plate soon reaches the temperature of 107°-110°.

For the ordinary staining reagents (in watery fluids) it is enough to place the air-dried preparation at about 110° C. for one half to two minutes. For differential staining mixtures, for instance the eosin-aurantia-nigrosin mixture, a time of two hours is necessary, or higher temperatures must be employed.

2. Chemical means.

a. To obtain a good triacid stain, the preparations may be hardened, according to Nikiforoff, in a mixture of absolute alcohol and ether of equal parts, for two hours. The beauty of specimens fixed by heat is however not quite fully reached by this method.

b. Absolute alcohol fixes dried specimens in five minutes sufficiently to stain them subsequently with Chenzinsky's fluid, or hæmatoxylin-eosin solution. It is an advantage in many cases, especially when rapid investigation is required, to boil the dried preparation in a test-tube in absolute alcohol for one minute.

c. Formalin in 1% alcoholic solution was first used by Benario for fixing blood preparations. The fixation is complete in one minute, and the granulations can be demonstrated. Benario recommends this method of fixing, especially for the hæmatoxylin-eosin staining.

These methods are described as the most suitable for blood-investigation in general. For special purposes, for instance, the demonstration of mitoses, blood platelets, etc., other hardening reagents may be used with advantage: Sublimate, osmic acid, Flemming's fluid, and so forth.

Staining methods may be classified according to the purpose to which they are adapted.

We use first those which are suitable for a simple general view. For this it is sufficient to use such solutions as stain hæmoglobin and nuclei simultaneously. (Hæmatoxylin-eosin, hæmatoxylin-orange).

Occasionally a stain is desirable which only brings out, but in a characteristic manner, a special kind of cell, e.g. the eosinophils, mast cells, or bacteria. Single staining is attained on the principle of maximal decoloration. (Cp. E. Westphal.)