SOME PATHOLOGICAL ASPECTS OF TUBERCULOUS MENINGITIS ... · practice to carry out a Pandy and Nonne-Apelt test, in an attempt to confirm whether the amount of protein present is pathological

202

SOME PATHOLOGICAL ASPECTS OF TUBERCULOUS MENINGITIS TREATED WITH STREPTOMYCIN

BY

Major G. B. S. ROBERTS Royal Army Medical Corps

IN treating tuberculous meningitis it is essential that an accur~te diagnosis be made as early as possible in the course of the disease. The early clinical signs which should make one suspect tuberculous meningitis have been described by Captain Clarke in his paper and he has stressed the importance of early examination of C.S.F. in all suspicious cases.' ,

While there are no cytological or chemical features in the C.S.F. which can 'be said to be absolutely diagnostic of tuberculous meningitis, yet the findings may be suggestive and enable a provisional diagnosis to be made subject to confirmation by finding the bacillus:

The following' are brief summaries of the methods used by this department :

CYTOLOGY

Many methods of performing C.S.F. cell counts are described and almost all of these are quite satisfactory provided they are carried out by the same operator to reduce personal error to a minimum. It is v,ery important that the count should be performed as soon as possible after withdrawal of the fluid from the

. body, normally within one hour. The most common error in doing C.S.F. cell counts is to count red cells as white and to obviate this it is considered advantageous in routine work to lyse the red cells by acid. As noted below, however, , separate red cell counts may be performed if required.

TECHNIQUE

The C.S.F. containers must be thoroughly shaken to obtain an even suspension of cells immediately before the fluid is withdrawn for the cell count. A white cell blood pipette is used and filled to the mark "1" with C.S.F. diluting fluid and then with C.S.F. to the mark "2." This is fixed and allowed to stand for three minutes to allow the cells to stain, then mixed again and a drop placed in the cbunting chamber in the normal fashion. It is advisable to use a chamber with a large area, e.g.

(I) An improved Neubauer. With this type of chamber if. the cells in the whole area, i.e. 9 sq. mm. are counted then the number of cells/cm. of C.S.F.= No. x J1\o x 'ior for practical purposes No. x i.

Alternatively, (II) A Fuchs Rosenthal chamber may be used. This has an area of 16 sq. mm. and a depth of 0·2 mm. No. of cells in whole area x rt x',? = cells per c.mm. = approximately No. x.;..

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C. B. S. Roberts 203

Alternatively, an area of 4'5 sq. mm., i.e. 4~ large squares, may be counted giving a direct reading. The combined stain and diluting fluid used is:

Crystal violet Acetic Acid Distilled water

0·5 c.c. 15·0 c.c.

250·0 c.c.

As acetic acid may be unreliable in producing lysis of red cells the following fluid may be used:

Brilliant cresyl blue Mj 1 hydrochloric acid

2 grm. \00 c.c.

Lysis of red cells by this method is very complete but it has the disadvantage that the staining of white cells is poor. It is only recommended if the number of red cells is very large. Red cells in C.S.F. may be very simply counted by a similar method except that the diluting fluid should be a simple stain with no acetic acid added. Toluidin blue (0·5 per cen~ aqueous solution) has proved very satisfactory.

If the total cell count is low differential cell counts are best done from the counting chamber while the total cell count is being done. Otherwise this may be done from stained dried films of centrifuged deposit.

CHLORIDES

A standard solution of silver nitrate is prepared containing 0·2907 per cent silver nitrate by weight. This is standardized against a I per cent solution of sodium chloride.

1 C.c. of C.S.F. is placed in a suitable container and diluted to about 5 C.c. with distilled water and a few drops .of a 10 per cent potassium cnromate added as an indicator. The silver solution is run in from a burette until the indicator changes to a rust red colour. If the silver nitrate solution is exactly 0·2907 per cent then each c.c. used in the titration is equivalent to 100 mg. NaCI per 100 c.c. of C.S.F.

PROTEIN

I C.c. of C.S.F. is placed ina small test tube and 0'2 C.c. of a 25 per cent trichloraq!tic acid solution is added. The resulting solution is compared with a series of standard opacity proteinometer tubes. The most satisfactory method of making a reading is to attempt to read print through the tubes. Readings are most difficult in the 40 to 60 mg. range and with ·such readings it is our practice to carry out a Pandy and Nonne-Apelt test, in an attempt to confirm whether the amount of protein present is pathological.

When the protein value exceeds 100mg. per cent the C.S.F. is first diluted with normal saline an appropriate number of times, and I C.c. of this diluted fluid is treated with 0·2 C.c. of trichloracetic acid as above, the reading is multiplied by the appropriate factor.




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204 Some Pathological Aspects of Tuberculous M eningiiis

SUGAR QUALITATIVE

Qualitative examination of C.S.F. for sugar gives little useful information and for this reason quantitative estimations of sugar have been done on all cases of suspected tuberculous meningitis.

Folin and Wu's method is sufficiently accurate and has been used here for some considerable time. .

The following is a summary of this method:

To I C.c. of C.S.F. add: 0·25 C.c. 2/3 N sulphuric acid. 0·25 C.c. 10 per cent ·sodium tungstate. 8·5 c.c. distilled water.

Allow to stand and filter. In a Folin and Wu's tube place

2 c.c. filtrate. 2 c.c. alkaline copper sulphate. (Folin and Wu)

Place in a boiling water bath for exactly six minutes. Cool rapidly for not more than two minute3.

Add 2 C.c. of phosphomolybdic acid and when bubbling has ceased make up to 12'5 C.c. with distilled water. Compare colour against known .standard. If a Lovibond comparator is used the figure read off is divided by 4.

Estimation of sugar in C.S.F. after the commencement of treatment with streptomycin has been found to be valueless as streptomycin itself is a strong reducing substance.

DETECTION OF TUBERCLE BACILLI

Containers for C.S.F. must be scrupulously clean as well as sterile. Rubber w',lshers from inside of metal screw caps should be boiled separately in distilled water and then refitted .. On no account must cork stopper be used.

In the identification of tubercle bacilli only organisms which are absolutely typical in shape, siZe, staining, etc., should be regarded .as "positive." Unless the suspected organism conforms in all. respects it should not be regarded as conclusive.

In almost all cases it has been possible to find tubercle bacilli on direct examination. Considerable. time, however, must be spent on the exploration of smears as the bacilli are often extremely scanty.

Part of the C.S.F. submitted is used for the tests outlined above and part is spun down in a centrifuge and smears are made froin the deposit. These are stained by Ziehl Neelsen's method, and searched for bacilli using a mechanical stage covering the slide from end to end. A useful economy is to do estimations of sugar and chloride on the supernatant fluid from the centrifuge tube.

A further portion of the C.S.F. is placed in the incubator overnight and examined the following day. This practice has been well worth while in our experience and has yielded a large number of positive results .. If any cobweb




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G. B. S. Roberts 205

clot forms in the specimen it should be carefully removed and spread on a slide . and examined. -

Culture of the C.S.F. was carried out in all cases. It is considered that to obtain consistent positive results the volume of the inoculum should be as large as possible. Two methods were used throughout.

(1) Centrifuged deposit or clot was inoculated on slopes of Lowenstein Jensen media, generally three slopes being inoculated' from: each specimen.

(2) A more satisfactory method was the use of a fluid medium-Kirschner's (Kirschner, O. (1932), Cent. Bakt., 124, 404)-being the one used here. This has the great advantage that large volumes of C.S.F. may be added to the prepared media. .

The method of preparation is

Sterilize.

Disodium hydrogen phosphate Potassium d1hyd1"ogen phosphate Magnesium sulphate Sodium sulphate ... Asparagine Glycerine Aqua destillata

3·p grm. 4·0 grm. 0·6 grm. 2·5 grriI. 5·0 grm.

20·0 grm. 1,000·0 grm.

Before use add 10 per cent sterile serum (human, horse, bovine or rabbit). Adjust reaction to a pH of 7·4. . Add penicillin to a concentration of 5 - 10 tll1its/ml.

Guinea-pig inoculation was also carried out in some cases but this method appeared to offer little advantage as the results with film and culture were so consistent. '

STREPTOMYCIN CONTENT

The streptomycin content of the C.S.F. was estimated at frequent intervals using a modification of Mitchison's diffusion method for which (the method is given as an appendix) fairly uniform C.S.F. streptomycin levels were found in all cases under treatment. Fig. 1 shows some 'of these results.

STREPTOMYCIN SENSITIVITY

The streptomycin sensitivity of the tubercle bacilli recovered from culture was estimated. In this series of cases the organisms all remained positive to streptomycin throughout the whole course of the treatment. In no case was it found that the C.S.F. yielded a growth of highly resistant tubercle bacilli.

DIFFERENTIAL DIAGNOSIS

It has been stressed above that the only certain method of diagnosing tuberculous meningitis is the demonstration of tubercle bacilli. A large variety of conditions produce a: lymphocytic response in the C.S.F. and these must all be considered .

. Tuberculoma, another type of intracranial tuberculosis, may closely simulate tuberculous meningitis. Every effort should be made, however, to differentiate the two lesions as the immediate prognosis of tuberculoma is good and spon"

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206 Some Pathological Aspects of Tuberculous Meningitis

taneous "cure" may occur. Although it may be silent and the C.S.F. normal for a long time, yet a leak may occur, and the cell count can rise very considerably and then fall slowly to normal without any treatment. At the same

. time tubercle bacilli mayor may not be found, a growth of tubercle bacilli was obtained from a single specimen of C.S.F. from the A.T.S. girl mentioned by CaptainClarke and it is thought probable that she has a tuberculoma.

Benign lymphocytic choriomeningitis is another lesion whi1:h closely simulates T.B. meningitis, but which undergoes spontaneous recovery. Accurate diagnosis of this condition is now possible by complement-fixation methods. In this condition the C.S.F. cell count is raised and predominantly lymphocytic. It is stated that the C.S.F. sugar does not fall as in T.B. meningitis but we have not sufficient cases to be able to express an opinion on this observation.

Poliomyelitis, cerebral abscess, encephalitis and even cerebral tumour may all give C.S.F.s practically identical with T.B. meningitis. The differentiation of these conditions is generally on clinical grounds.

CEREBROSPINAL FLUID IN THE EARLY STAGES OF THE DISEASE

The fluid is usually under increased pressure. Manometric readings usually being between 250 and 300 mm. of water. The fluid comes freely from the needle and 10 to 15 c.c. may be withdrawn with safety. .

The fluid is generally crystal clear. The cobweb clot described in so many textbooks is not often seen in the early stages, and its formation appears to depend directly upon the amount of protein present. A clot, however, may sometimes form if 'the fluid is allowed to stand overnight in the incubator.

The cell count is increased averaging 300 cells per c.mm'., with extreme values ranging from 80 to 600. The majority of the cells present are lymphocytic with 25 to 5 per cent of polymorphs.

Protein content is also increased the actual figures generally being between 100 and 150 mg. per cent. ' .

Chlorides are diminished, 640 mg. per cent being the average level in the initial stages, in our cases, glucose content too is diminished-negative reaction generally being obtained when a quantitative BeIiedicts was performed. If estimated quantitatively the sugar content is found to be about 40 mg. per cent. - 1£ the case is kept under observation the chloride and sugar content of the

C.S.F. show a tendency to fall. In one of our cases the sugar content was found to be 50, 35, 30 mg. per cent on three successive examinations. It is valueless to carry out sugar estimations in the C.S.F. once streptomycin therapy has ,been commenced, as the drug itself is a strong reducing agent.

CHANGES IN C.S.F. IN CASES UNDER TREATMENT

The most striking feature in cases of T.B. meningitis is the very marked variation which occurs in the C.S.F. in a short space of time--changes which apparently are unrelated 'to any change in the patient's clinical condition. While the cell count shows the greatest variation, the protein content also shows wide fluctuation. The chloride content on the other hand remains relatively constant over a period showing only a slow rise or fall.




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G. B. S. Roberts 207

In general terms it can be said that once treatment has been commenced the cell count continues to rise for two or three weeks, sometimes reaching a fairly high figure and then falls steadily. The protein too may show a similar rise followed by a slow ·fall. The protein content returning to normal more slowly than the cells. The chloride content in our successful cases has shown a slow rise to normal. These results are shown graphically in figs. 1 and 2.

Tubercle bacilli can generally be found either by smear or culture in the C.S.F. for one to three weeks. Its persistence after this time or its reappearance is regarded as a bad prognostic sign.

The following is a brief summary of the findings at post-mortem in the fatal cases in this series. '

Case 3.-The right lung showed a healed primary subpleural focus with calcification of the hilar lymph nodes. This, lung was firmly bound to the chest wall by diffuse dense fibrous adhesions, the appearance being that of a' healed tuberculous pleurisy. In addition the patient had a tuberculous left knee and had a large tuberculous mass in his

800

700

60 CHLORIDES PROTEIN Mgm.% 500

CELLS percmm.400

300

200

CASE 4. DIED

t ~CHL6RIDES

~ \\ .

~ ..... :,," .... ,

800

700

600 CHLORIDES PROTEIN Mgm.% 500

CELLS 0 pertmm 4

300

200

CASE 6.

/~ DIED 0" I

" , l'

~/\ • I

: . I CHLORIDES I I . I I I , I I I I I \ I I

e---8,,' ~ \: .... - \ \!:... .....•.. .... .CELLS :\' 'e' " . ........ '." 100 ••... ·•·•· •.. ·· .. , ....• PROTE.IN

" 100 STREPTOMYCIN'

.... . .... 0 .. ··.• .PROTEIN

STREPTOMYCIN • CELLS

J gl~~~~~~JTS L-JiiiL..r.ili!~~W!-4'"""'"5~"l!6~""'7-.",a-"'9~IO W E.EKS

10\~9~TuE~~swm....~-l::---'!i1!:-~-IiiI~~-;;8-9n';nIO

FIG. I (Cases 4 alld6).~Fatal cases. The graphs show the cells, protein, chloride and streptomycin content of the C.S.F. under treatment.

800

700

600 CHLORIDES

PROTEIN.' 500 mgm.%

CELLS 400 percmm, 300

200

100

CASE 2

,. -.... -CHLORIDES ~ !~ •••• e· •• • .~ •• .................... 'rf"'.

I • I • ....... : \

'I • .. ..... \. "0" .• .. \

•.••.•••••• \ • .11 ....... • ' .............. -.. ••• -0-•.•

.... " - "'0.. .......... ....... PROTEI N ...... -....... ....... ..... .... .,., ............... . ......... -................. ............. ..... • .. CELLS

I 2 3 4 5 6 7 B 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 2S 26 27 28 29 30' WEEKS

FlG. 2 (Case 2).-This case did well and was eventually disoharged apparently cured. The graph shows the slow rise of chlorides to normal and the gradual fall of cells and protein. The sharp rise in the cell count after about four weeks' tJ:eatmem has occurred in a number of our cases and by itself does not appear to ,be a bad prognostic sign.

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208 Some Pathological Aspects of T"berClllolI s l\1C/li1!gitis

m.esen tery. Th is had given rise to tuberculo us peritonitis. A small t uberculoma was present in the cerebellum. This has become adherent to the dura . Signs of tuberculous meningi ti s were minimal at the base of the brain, but vcry gross tuberculous involvement of the spinal meninges was present (sec fig. 3).

FIG. 3 (Case 3).- This shows a porlion o( the spinal cord. J\bund ant large tubercles ale present coalescing o n onc area. A considerahle amount of ha::morrhagc is present.

On section of the brain h:.cmorrhagc into the d ilated lateral vcnrride was seen. Thi s case is an example of a healed prilnar)' tuherculous Jesion, with many secondary

tuberculous lesions-;lmee, peritonitis and brain. E!'cape of infected material from the cerebellar tuberculoma is considered ro he nh e ca use of the meningitis.

Case 4.-1n this case all active subpleural lesion wir.h large cascu lIs tuberculous bronchial lymph nodes was found (see fi g:. 4). A seamy mil iary spread is seen in lungs and spleen. The base of the bra in was cnvered wi th a very dense fibrou s membrane, particularly around the optic chiasma. Small tubercles are seen along the cerebral vessels, and a small tuberculoma is present on the right middle cerebral artery (fig. 5). On sect ion the brain shows gross ilHernal hydrocer~halus.

This case is an example of a primary tuberculous infecrion in a young adult which has progressed to miliary spread of tubercle bacilli. These have sculed predominantly on the meninges and caused meningitis.

This type of ca se would offer least chance of successful treatment as th e pali elH'~

resistance of the spread o[ tubercle bacilli would appear to be low. Case 5.- This case showed an aCLive pulmonary lesion with caseous mediast inal lymph

nodes. A \'cry gross miliary spread was present to all organ~ in tht: body including the brain.

Case 6.~Jn this case thc-re were abundant tuocrcles both on trhe vertex and basal surfaces of !fhe hrain and a co nsiderable amount of fi·brous ex uda te was present , in the interpeduncular fossa, Sylvian fissu res and mid -brain. The cerebral convolutions showed marked flattening and on sec tion internal hydrocephalu s wa s noted .

Ko primary site of the tuberculous infection was found.

ACKXOWLEDGIVIEKTS

I wish to thank Dr. R. L. Vollulll of the Radcliffe Infirmary. Oxford. for




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c. B. S. Roberts 209

FIG. 4 (Case 4).- This view of the lung shows caseolls tuberculous hila lymph nodes rdated to the subpleural Jesion.

carrying out streptomycin estimations on these cases and advice on many problems on this subject.

APPENDIX l\1ITCIIlSON'S DIFFUSto~ l\1ETHOD FOR ASSAY OF STl~EPTOMYCIN

Principle.- Strcptomycin diffuses t\hrough a staphylococcus-inoculated agar causing a clearly marked zone of inhibition afrel' eighteen hours' incubation. The ZOIlt! of inhibition is proportional to the concentration of streptomycin.

Summary of Method.-A special diffusion agar is melted and cooled to 45 0 C. and then inoculated with an optimum amount of staphylococcus cuhure. The nutrient agar is in 19 mil. amounts in 1 oz. wide-necked bottles. For inoculum 0'4 ml. of an o\'ernight broth culture of s(aphylocoCCllS is added to 10 ml. of !4 strength saline, mixed, and I ml. of the suspension is added to the bottle of agar, which is then shaken vigorously for half a minute. Place the bottle in a 45 " C. water hath, and wait for a minute to allow the larger bubbles of air to rise to the top of the medium. The agar is then pipened into 3 mill. diameter tubes, to form a column 2 cm, deop, The tubes should he kept in a rack which will keep them vertical throughout the test. When the agar has solidified rhe streptomycin standard solutions and unknowns <Ire pi petted on to the surface of the agar. The depth of the fluid is immaterial, except that it should be greater than I mm. The rack is incubated at 37° C. for eightt!en hours and the zones of inhibition measured.

For measuring the zone of diffusion use a microscope equipped with a % ohjective, No. 2 ocular fitted with a hair-line, and a mechanical -stage fitted with a Vernier scale. :'vIount the diffusion tube in a cradle of plasticine on a slide, and measure the distance from the agar meniscus to the foremost and largest demarcation line of the staphylococcus colonies.

Calculations ......... The usual preliminary hefore adopting the test i!'; to set up in triplicate




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210 Some Pathological Aspects of Tuberculous Meningitis

FIG. 5 (Case -+).- This view of the base of the brain shows the optic nerves hUllnd in Vt!ll' dense fibrous tisSlle. A small tuberculoma is presclIl un lhe right middle cerebral artery just after its commencement. Tubercles MC st.:;:wty hut may be seen near Ihe olfactory nerves.

falling concentrations of streptomycin, namely 64 , 32, 16, 8, 4, 2, units per ml. The zones of diffusion arc accurately measured to the first place:! of decimals. The 3\"Cragc of the three readings is squared and the figure is plorted against the logarithm of the concenTratiun of streptomycin. For concentrations between 2 and 64 units pcr mL the points should fall on a straight linc. If tbey do nor fall on a straight line there is some errOr in the method. Once linearity has been achieved only three standards need be lISed 6-1, 16 and 2 units per mL The standard curves made froIU day LO day should be parallel or nearly so.

ft must be ernphasil:ed thaL the srandal·ds shollld be set up rogethcr with the unknowns, for each boule of agar used. The graph is dtawn from the standard read ings, 'lnd lhe squares of the unknowns arc read off as logarit.hms of the cuncentratiun of sLreptomycin pe,r m\., then converted LO units per ml.

The zone of inhibition of growth is not directly proportional to the streptomycin concentration, e.g. the difference between 2 and 8 Ltnits is 2·6 mm.; and the difference hetween 32 <Ind 64 units is 0-6 mm.; thence it is realized tbar the range below 64 units is the more accurate for tlIe conversion of difl"usion to the cunCentf.:llion of streptomycin. Any unknowns that are greater than 64 units should be diluted in phosphate buffer pH 7·8 accordingly.

Agar COllce11tratioll.-The ideal is to experiment 'ViIh a batch of agar powder and discover tbe concentration which gives the greater dill"crcncc between the di ITusions of 2 and 64 units. The final percentage recommended in Mitchison's paper is 1·25 per ccnt;




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C. B. S. Roberts 211

this concentration with my agar allows a comparatively poor diffusion compared with a I per cent concentration, 'Which gives a wider range of diffusion; and of course the weaker the agar the better the growth of the staphylococcus so that demarcation is facilitated in the deeper, more anaerobic zones, which in a 1·25 per cent concentration are scattered and the colonies are exceedingly small.

Thus with the higher concentration of agar there is slower diffusion of streptomycin so that staphylococcal colonies grow to, a visible size before an inhibitory concentration of streptomycin reaches them. The depth of diffusion can be increased and the line of demarcation improved by refrigerating the tests, for two hours before incubating them; but when the optimum concentration of agar has been found, this is not necessary, as the rapid diffusion reduces scatter of colonies to 'a minimum.

Optimum pR.-The op~imum pH for diffusion and antibiotic activity of streptomycin is, apparently 7·8, ana this critical pH is necessary for the medium and buffer used for diluting the standard 1 per cent streptomycin, urine; etc.; C.S.F. and serum are usually alkaline enough without adjustment of the pH. Cresol red is a useful indicator for this particular pH range.

Diffusion Agar.-2·5 per cent nutriep.t agar-l volume, this must be clear and free from phosphate or other precipitate. 2 -per cent Evan's O£ Bacto peptone in distilled water-I volume, adjust pH to 7·8 with cresol red as indicator using the tile method. i.e. a change from yellow to a dirty red 'colour.

For trial purposes increasing amounts of peptone water can be added to the agar volumes to estimate the optimum agar conc. '

An indicator can be i:n,cluded in the medium without detrimental effects. The final agar is bottled in 19 rnl. amounts and autoclaved at IO lb. for fifteen

minutes. Streptomycin-sensitive Staphylococcus.-Most strains o( Staphylococcus pyogenes are

sensitive to streptomycin. 'A standard strain can be obtained from the eolHndale Laboratories.

Streptomycin Standard.-A I per cent solution of streptomycin is issued from Collindale every three months and all dilutions for use as standards for diffusion technique should be made by accurate pipetting using phosphate buffer.

Diameter of Glass Tubing.-Various diameters of glass tubing were tested ,in triplicate with the same concentration of streptomycin, and no discrepancy of zones was obs~rved. The range of diameters was from 3 mm. through intervals of 0·1 mm. to 3'7 mm.

Concentrations Below 2 Units per ml.-Streptomycin concentrations between 0·5 and 2 units/ml. can easily be assayed by including 0'5, 1'5, unit standards with the usual set. A separate graph is made plotting the actual units of streptomycin against the square of the readings of the diffusion zone. The unknowns are then read as units perml.The graph 'shows linear tendencies up to 4 units per ml.

Error of Method.-Mitchison claims that error up to 1'00 units per ml. to be 20 per cent. I have found an error of only 8'5 per cent up to 64 units per ml.

Technical Points Not Mentioned Above.-(I) Ensure that the agar has been cooled to 45° C. before adding staph. suspension and do. not keep at this temperature for more than five minutes once inoculated.

(2) Withdraw Pasteur pipette slowly through foam on agar, otherwise a quick movement will cause foam to attach to pipette.

(3) Use plain heart broth, for overnight culture of s,taphylococcus. Glucose broth reduces the number of viable bacteria due to acidity of fermentation.

(4) A wide bore pipette should be used to facilitate quick discharge of the medium into the tubes.

(5) The diffusion tubes are 3 x 60 mm.; sealed, at one end. They are cleaned by washing out the agar by using a pipette and tubing attached to a water tap, followed by swabbing with a wire swab, washed out again with tap water and then distilled water. Shake out excess of water, invert in a beaker, to dry. Sterilize in a tin in hot air oven. The tubes are not plugged.




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SOME PATHOLOGICAL ASPECTS OF TUBERCULOUS MENINGITIS ... · practice to carry out a Pandy and Nonne-Apelt test, in an attempt to confirm whether the amount of protein present is pathological