The following are the most useful procedures for examining spinal fluid for psychiatric diagnosis: (a) Cell count. (6) Lange's colloidal gold test, (c) Special protein tests (Noguchi, Ross-Jones, Pandy). (d) Wassermann reaction (described in Appendix II).

(a) The cell count must be done immediately after the fluid has been collected, as the cells soon undergo autolytic action outside of the body.

The following equipment is required: (1) Mixing pipette like that used for making white blood-corpuscle counts. (2) Fuchs-Rosenthal counting chamber, ruled as illustrated in Fig. 7.1 (3) Clinical microscope, preferably with mechanical stage. (4) The following staining solution:

Methyl-violet....................

0.2 gram

Acetic acid.......................

4.0 c.c.

Distilled water...................

96.0 c.c.

Shake well and filter before using.

The staining solution is drawn into the pipette up to mark 1, and then the spinal fluid, after being thoroughly shaken to insure uniform suspension of the cells, up to mark 11; the pipette is then shaken for about five minutes to mix the stain thoroughly with the fluid.

As that part of the fluid which is in the stem of the pipette does not become mixed with that in the bulb, and is drained off before a drop is taken out for the counting chamber, the dilution in the bulb, in calculating the results, is to be considered as in the proportion of 9 parts of spinal fluid to 1 of the staining solution.

1 Excellent counting chambers of American manufacture are to be had from Max Levy, Philadelphia.

After draining off the fluid in the stem of the pipette - three drops - a drop of suitable size is placed in the counting chamber and a cover glass put on; or, still better, if one of the new counting chambers of American manufacture is used, a drop is allowed to flow in by capillary attraction. It is best to let the fluid stand in the counting chamber about a minute before the counting is begun; this permits the cells to settle on the bottom so as to be as nearly as possible in the same focus as the ruling of the chamber.

Lumbar Puncture Cell Count Chemical Tests Part 2 14

Fig. 7. - Ruling of Fuchs-Rosenthal Counting Chamber.

The count is made most conveniently under rather low magnifying power of the microscope, say, 16 mm. objective, 10 x eye-piece, Bausch & Lomb.

The dimensions of the counting chamber are 4 mm. on each side and 0.2 mm. in depth, i.e., 3.2 cu.mm. As but nine-tenths of the mixture in the counting chamber is spinal fluid, the remaining one-tenth being staining solution, all the cells counted in one chamber represent the cell content of 2.88 cu.mm. of spinal fluid. It is customary to express the findings in number of cells per cubic millimeter of spinal fluid; and this is derived, of course, by dividing the total number of cells counted over the entire ruled area of a Fuchs-Rosenthal chamber by 2.88.

It is advisable to make two or three counts with different drops and to report the calculated average rather than the result of a single count.

The number of cells per cu.mm. of spinal fluid varies considerably both in health and disease, and there is no definite point of demarcation between the two. Most pathologists consider any number under 5 as a negative finding, between 5 and 8 as doubtful, and over 8 as positive.

The staining solution, for which the formula is given above, will enable one to differentiate between white and red corpuscles. The small mononuclear elements assume a deep blue color with a narrow lighter rim of cytoplasm. Red cells appear light colored, hyalin, translucent. Polymorphonuclear elements are recognized by their nuclei. It would be easy to dissolve all red cells by adding more acetic acid to the staining solution. We have purposely not done this, as it is an advantage to be able to count red cells as well as white ones and thus have a measure of the contamination with blood that there might be. If more than 20 red cells per cu.mm. are found, the cell count as well as the colloidal gold and other protein tests are not to be relied on.

In cases in which clinical data would lead the physician to expect a positive finding, while the reported finding is either doubtful or negative, the lumbar puncture may be repeated at the end of ten days. Either on first or second examination almost all cases of general paralysis and cerebral syphilis furnish positive findings, while most other psychoses furnish negative ones. In all acute infections of the meninges polymorphonuclear cells are found.

(6) Lange's Colloidal Gold Test.1 - The reagent is prepared as follows: One uses water which has been thrice distilled, and, in being distilled, has not been allowed to come in contact with rubber connections, all connections of the distilling apparatus being of cork which has been well boiled beforehand. An apparatus like that described by Miller, Brush, Hammers, and Felton2 is very useful. Three hundred c.c. of such thrice-distilled water is put into a beaker of Pyrex or Jena glass, and the beaker is placed on wire gauze over a hot flame. When the water has reached the temperature of 60° C. 3 c.c. of a 1% solution of gold chloride in similarly thrice-distilled water is added. Following this 2.1 c.c. of a 2% solution of potassium carbonate of the highest purity is added. The solution is then brought quickly to 90° C. At this point 2.1 c.c. of a 1% solution of formaldehyde is added gradually while stirring. This is prepared by diluting 1 c.c. of commercial formaldehyde solution (40%) with 39 c.c. of thrice-distilled water. The whole is kept at a temperature of 90° C. until a pink tinge appears; the beaker is then removed from the flame and the reaction allowed to complete itself. It should not be allowed to boil violently at any time, but may bubble gently.

The solution thus prepared should be perfectly clear and without a bluish tinge. It will keep for weeks or months.

Before using the solution is tested as follows: 5 c.c. are put into each of three small test-tubes; then 0.5 c.c. of a 1% sodium chloride solution is added to the first tube, 1.0 c.c. to the second, and 1.7 c.c. to the third. The first tube should show no change in color at the end of an hour, the second should assume a blue tinge, and the third should show complete precipitation with colorless supernatant fluid.