The blood may be examined by means of films, stained or unstained, or by sections, celloidin or paraffin.
A. FILMS. The blood may be obtained from the pulp of the ring finger, from the skin over the knuckles, or from the posterior aspect of the lobe of the ear. The place selected should be carefully cleansed with water, soap and 1/1000 mercuric chloride solution, and finally with alcohol and ether. A puncture is made with a sterilized triangular needle or knife, or a stub-pen with one point broken off. The last-named makes a most useful and inexpensive instrument for this purpose. The puncture should be made by a quick and deep stab, so that sufficient blood can be obtained from one stab-wound. Pressure should not be employed to force blood from the wound. Bleeding may be encouraged by letting the arm hang down, or by applying pressure in the furrow of the terminal joint of the finger. The first drop of blood should be wiped away with a clean towel. When the second drop reaches the size of a pin-head touch it with the under side of a perfectly clean cover-glass, held by forceps, not by the fingers; place this cover-glass immediately upon another clean cover, so that the blood will spread out between the two covers in a thin film. The covers are then separated by sliding them apart without pressing or squeezing; place covers with film side upward, and dry in the air. The films should not be touched with the hands; forceps alone should be used to handle them. If the blood does not dry as quickly as it is spread the film will be too thick. Films may be made upon slides in the same way, or the drop of blood may be caught upon the edge of a clean cover, slide or “spreader” and then drawn rapidly across a slide. The dried film may be marked by scratching with a needle-point the number and date on the film itself. Blood-films may be fixed without drying by exposure to the vapor of formol or osmic acid for several seconds and then dropping into absolute alcohol. Formol alcohol, saturated mercuric chloride solution or Flemming’s solution may also be used for the fixation of wet films, fixing for 5-10 minutes, and washing thoroughly after each of the last two solutions. The dried film may be fixed by exposure to heat (110-115°C.) for 5-10 minutes for Ehrlich’s triple stain, and for 2 hours for the methylene-blue-eosin methods; 30-60 seconds at a temperature of 120°C. may suffice; the film should be brought at once into the required temperature. Heat-fixed films are improved by dipping them for a few minutes in mercuric chloride solution and then washing well before staining. Acetone-free methyl alcohol (1-2 minutes), absolute alcohol and ether in equal parts (½-12 hours), formol-alcohol (1-2 minutes), alcoholic mercuric chloride (absolute alcohol 25 cc., ether 25 cc., 5 drops of a 2 grms. mercuric chloride solution in 10 cc. of absolute alcohol) for 2-5 minutes, and formol-vapor are the chief solutions used for fixing the air-dried film. For ordinary work methyl alcohol, formol alcohol, and the absolute-alcohol and ether mixture give good results; heat fixation brings out the granules well, and mercuric chloride is a good fixative for the leukocytes. The combination of fixation and staining, as in Leishman’s or Wright’s modification of the Romanowsky method, is also recommended for general work.
For the staining of blood-films an almost endless variety of staining-methods can be found in the literature. Many of these represent slight deviations in the method of making the stain or in its application, such deviations marking stages of improvement in the development of the method. It is not necessary, therefore, to give all of these methods, but to consider only the latest modifications of value. In a general way blood-stains may be divided into five classes:—
1. HAEMATOXYLIN AND EOSIN.
Fix in equal parts of absolute alcohol and ether for at least 30 minutes; stain with hæmalum and eosin, or Ehrlich’s acid hæmatoxylin and eosin. By adding O.5 grm. of eosin to the formula for Ehrlich’s acid hæmatoxylin a combination stain can be made that is very good for blood-films fixed by heat or absolute alcohol and ether. Stain 2-24 hours, wash, dry and mount in xylol balsam.
2. EOSIN AND METHYLENE-BLUE.
Fix by formol (dried film over 40 per cent formol for 1 minute); absolute alcohol for 1 minute; stain 5 minutes in a 1 per cent watery eosin; then without removing eosin place in watery methylene-blue for 2 minutes; wash quickly; dry in air; balsam.
3. MIXTURES OF EOSIN AND METHYLENE-BLUE.
The numerous mixtures of methylene-blue and eosin are not very stable, can be kept for a few days only, and give varying results. Jenner improved this method of staining greatly by collecting the precipitate formed by the addition of eosin to methylene-blue, and dissolving it in pure methyl alcohol, thus giving a solution that fixes and stains at the same time. The May-Grünwald method is practically the same.
Jenner’s Method.