MIDDLEBROOK-DUBOS HAEMAGGLUTINATION …J. clin. Path,. (1953), 6, 64. THE MIDDLEBROOK-DUBOS...

J. clin. Path,. (1953), 6, 64.

THE MIDDLEBROOK-DUBOS HAEMAGGLUTINATIONTEST AND ITS HAEMOLYTIC MODIFICATION IN

THE DIAGNOSIS OF TUBERCULOSISBY

J. C. SHERRISFrom the Bacteriology Department, Radcliffe Infirmary, Oxford

(RECEIVED FOR PUBLICATION SEPTEMBER 30, 1952)

The detection of serum antibodies to M. tuber-culosis by the agglutination of sheep's red cellspreviously sensitized with an aqueous extract ofphenol-treated bacilli of the H 37/Rv strain wasfirst reported by Middlebrook and Dubos (1948).The short series of tests using human and rabbitsera reported by these authors suggested that thereaction was specific. They showed also that theactivity of the antigenic extract was associated withthe polysaccharide fraction and that a sample ofdeglycerinated old tuberculin was equally capableof acting as the sensitizing agent. These findingswere confirmed and extended by Gernez-Rieux andTacquet (1949, 1950a and b) and by Sievers,Ulstrup, and Winblad (1950), using the samemethods and antigenic extract as Middlebrookand Dubos.Many modifications of the test have been de-

scribed. Scott and Smith (1950) used old tuber-culin (Lederle) of four times standard strength asthe sensitizing agent, and Rothbard, Dooneief, andHite (1950), Smith and Scott (1950), Fleming,Runyon, and Cummings (1951), and Kirby, Bur-nell, and O'Leary (1951) have reported series oftests, using this method. Adcock, Haley, andDavey (1951) and Hinson, Jones, and Chamberlin(1952) used the same antigen, but replaced thesheep cells by human Group 0 cells. Sohier,Juillard, and Trimberger (1950) used the purifiedtuberculin prepared by the Pasteur Institute as thesensitizing antigen, and this was compared withthe original extract of Middlebrook and Dubos byGernez-Rieux and Tacquet (1950a and b), whofound a close correspondence between the resultsobtained. Further modifications were described bySohier, Trimberger, and Juillard (1950) using humanGroup 0 Rh-negative cells, the purified tuberculinof the Pasteur Institute as the sensitizing antigen,and a shorter test period. Fewer false positiveresults were claimed by this technique. Boyden(1951) described a test using Weybridge purifiedprotein derivative (P.P.D.) as the sensitizing agent,

and Hilson and Elek (1951) and Thalhimer andRowe (1951) both described micro-methods for thetest, using human Group 0 cells.Haemolytic modifications of the test in which

guinea-pig complement was added to the reactingsystem and lysis occurred in the presence ofspecific antibody were described by Fisher andKeogh (1950), by Thomas and Mennie (1950), andby Middlebrook (1950). Discrepancies betweenthe results of the haemagglutination and the lysistests were found both by Middlebrook (1950) andby Mollov and Kott (1952) and the possible exis-tence of two antibodies was postulated.

Considerable differences in the specificity of thetests have been reported both with the variousmodifications that have been described and wherecomparable methods have been employed, and theinvestigation reported here was designed to com-pare the haemagglutination and lysis tests, to assessthe possible clinical value of the results, and togain some further information as to the effect ofvarying the conditions and reagents. The materialinvestigated consisted of human sera from healthysubjects, from cases of tuberculosis, and from somepatients suffering from diseases other than tuber-culosis. The techniques used throughout the in-vestigation required only small quantities of sera,and it was thus possible to perform several testson each serum.

The Haemagglutination TestThe methods described are essentially those of

Middlebrook and Dubos (1948), except for the use ofdialysed tuberculin as the sensitizing antigen and ofsmall reagent volumes for the test.

Reagents.-The following reagents were used.

Phosphate-buffered Saline.-Eagle's buffered saline,pH 7.4 (Eagle, 1937), was used throughout. It con-sists of

NaCl ..

KH2PO4Na2HPO4 (anhydrous)Distilled water to 1 litre.

170g.27g.

.11-3g.

on April 22, 2020 by guest. P

rotected by copyright.http://jcp.bm

j.com/

J Clin P

athol: first published as 10.1136/jcp.6.1.64 on 1 February 1953. D

ownloaded from

http://jcp.bmj.com/

THE MIDDLEBROOK-DUBOS HAEMAGGLUTINATION TEST

The stock solution was autoclaved at 5 lb. pressurefor 15 minutes and diluted 1 in 20 with sterile distilledwater before use.

Sheep's Blood.-One volume of sheep's blood wascollected aseptically into 1.2 volumes of modifiedAlsever solution, and stored at 4° C. The bloodwas kept for three days before use and was rejectedafter one month.Modified Alsever SolutionGlucose. 410 g.Sodium citrate.1-60 g.Sodium chloride.0-84 g.

Distilled water.200 ml.The pH was adjusted to 6.1 with 10% citric acid

and the solution sterilized in the autoclave at 10 lb.pressure for 10 minutes.Old Tuberculin.-Burroughs Wellcome old tuber-

culin was used throughout as the sensitizing agentin testing routine sera. It was deglycerinated, freedfrom phenol, and rendered isotonic by dialysing in5-ml. amounts against three 4-litre changes of nor-mal saline over a period of 36 hours. The dialysatewas always found to be approximately three timesthe original volume of old tuberculin. This prepara-tion was stored at - 100 C., and under these condi-tions it maintained its activity for at least a month.Sera.-Sera were pipetted off on receipt in the

laboratory and immediately frozen and maintainedat - 100 C. Absorbed sera were similarly stored atthis temperature.

Test Tubes.-Durham tubes, 6 x 45 mm., were usedfor the test. They were sterilized in the hot-air ovenat 160° C. for one hour.Methods.-These were as follows.A bsorption of Sera.-Heterophil antibodies to

sheep cells were absorbed out by the method ofMiddlebrook and Dubos (1948). The unabsorbedsera were thawed at room temperature and diluted withan equal volume of phosphate-buffered saline. Com-plement was inactivated by heating the diluted serumin a water-bath at 56° C. for 30 minutes. To 2 ml.of this inactivated 1-in-2 dilution of serum was added0.2 ml. of washed packed sheep cells and even suspen-sion ensured by vigorous shaking. The mixture wasallowed to stand at room temperature for 10 minutesand the cells then deposited at 2,500 r.p.m. in anangle centrifuge. Without removing the supernatantanother 0.2 ml. of packed cells was added and evenlysuspended, using a Pasteur pipette, care being takennot to resuspend the deposited cells. After a further10 minutes at room temperature the cells were spunoff in the angle centrifuge and the supematant re-moved as in a l-in-2 dilution of absorbed serum.

Sensitization of Sheep's Cells.-Sheep's cells pre-viously stored in Alsever solution were washed threetimes in at least 10 volumes of phosphate bufferedsaline in an angle-head centrifuge. After the lastwashing the cells were well packed down and as muchof the supernatant fluid as possible was removed.One volume of packed cells was added to 49 volumes

E

of dialysed old tuberculin, well mixed, incubated in awater-bath at 370 C. for two hours, and shaken every15 minutes. The cells thus sensitized were againwashed three times and finally resuspended in phos-phate buffered saline to give a concentration of0.25% for the haemagglutination test. A controlsuspension of 0.25% unsensitized cells was also made.The suspensions were stored at 40 C. and the sensi-tized cells were found to be fully agglutinable forabout four days, after which time lysis began to beapparent. In practice, however, the cells were sensi-tized the day before testing the batches of sera.The Tests-The reagents were added by a dropping

technique, using Pasteur pipettes calibrated to deliver0.02 ml. of water in each drop. The method andvolumes (as drops) used in the haemagglutinationtest are shown in Table I. Serum and antigen con-

TABLE ITHE HAEMAGGLUTINATION TEST

Tubes

Reagents Serum Anti-123 4 5 ~Con- gen

trot Conltrol

Phosphate-bufferedsaline.. .. 2 3 - 2 3 2 4

Serum I in2 .. 2 1 - - - 2 -

SerumI in 16 - - 4 2 1 - -Sensitized cells .. 4 4 4 4 4 - 4Unsensitized cells .. _ 4 -Serum dilution* .. 1/4 1/8 1/16 1/32 1/64 1/4

* Titres throughout this paper refer to initial serum dilutions anddiscount the added cell suspensions.

trols were included and a positive serum of knowntitre and a negative serum were put up with eachbatch of tests. The contents of the tubes were wellmixed by shaking and the tests incubated at 37° C.in the air incubator for two hours. The tubes werethen again shaken vigorously to resuspend depositedcells and allowed to stand overnight at roomtemperature.

Reading the Tests.-The tests were read by gentlytapping the tubes with the forefinger until thedeposited cells were resuspended. Where agglutina-tion had taken place the cell clumps were disturbedreadily, often in a single mass, and the end-point wastaken as the last tube showing definite agglutinatedclumps as seen by the naked eye. Continued agita-tion of positive tests readily breaks up the agglutina-tion, and care should be taken to tap the tubes onlysufficiently to disturb the deposit. Tests were judgedas negative when the deposited cells resuspended lessreadily on tapping and no agglutinated clumps wereseen. Attempts to read the tests according to depositpatterns were abandoned, as it was soon found thatagglutinated cells occasionally sedimented as a sharplydefined button of cells indistinguishable from thedeposit in negative and control tubes.

Repeated tests on the control positive serum andon some test sera showed the method to give results

65



j.com/

J Clin P


ownloaded from

http://jcp.bmj.com/

J. C. SHERRIS

which were reproducible to within one tube of theoriginal reading.

The Lysis TestThe preparation of sera and the sensitization of

the cells were carried out in the same way as for thehaemagglutination test, a 0.5% suspension of sensi-tized cells being used in place of the 0.25% suspen-sion. The same reagents and test tubes were used.

Preparation of Complement.-Lyophilized guinea-pig complement (Sharp and Dohme) was reconsti-tuted and diluted 1 in 3 with phosphate-bufferedsaline. One part of washed packed sheep's red cellswas added to 15 parts of diluted complement. Themixture was allowed to stand for 10 minutes at roomtemperature and the cells deposited at 2,500 r.p.m.in the angle centrifuge. The procedure was repeatedwithout removing the supernatant, care being takennot to disturb the deposited cells. The supernatantwas finally removed as a 1-in-3 dilution of absorbedcomplement. It was used within two or three hoursand kept meanwhile at 4' C. This method is identi-cal with that described by Middlebrook (1950), exceptthat absorption was carried out at room and not atrefrigerator temperature.Complement must be of high titre. The amount

used in this test represents an excess, and a check onthe activity of the complement is given by the haemo-lytic titre of the standard positive control serumincluded with all batches of tests. Consistentlyreproducible results were obtained in practice.The Test.-The tests were set up by the dropping

technique. The method and volumes were the sameas for the haemagglutination test except that onedrop of the absorbed complement was added to eachtube. Similar controls were also included. The rackswere placed in an air incubator at 370 C. for onehour and were well shaken both before incubationand at 15-minute intervals. On removal from theincubator the tests were read and an arbitrarilyjudged 50% haemolysis was taken as the end-point.There was no difficulty in determining the haemo-lytic titres, as in all cases the end-point was sharplydefined and in the vast majority of positive testshaemolysis was complete in all but the last tube.As with the haemagglutination test the results werefound to be reproducible to within one tube of theoriginal reading. After storage for several monthsat - 100 C., however, some sera showed a definitereduction of lytic activity.

Effects of Modifying the Reagents and Con-ditions of the Tests

The Red CelIs.-Sheep's blood preserved inAlsever solution was compared with defibrinatedsheep's blood for the haemagglutination test. Thedegree of agglutination was most marked using"Alsever"' cells and the titres were usually one

tube higher than with defibrinated blood. In com-

parative experiments cells from defibrinated blood

were found to be as effective as those from bloodin Alsever solution for absorbing the heterophilantibodies from the sera.Comparisons were made for both the haem-

agglutination and lysis tests, using sheep cells andhuman Group 0 Rh-negative cells. The humanblood was preserved in Alsever solution or in theacid-citrate-dextrose solution used for preservingblood for transfusion (Mollison, 1951). The con-ditions of washing and sensitizing the human cellswere those described for sheep cells. The seraused for comparative studies were divided intotwo parts. Both parts were diluted 1 in 2 and in-activated at 56' C. for 30 minutes, but only theportion used for testing with sheep cells wasabsorbed. Comparative tests were performedsimultaneously on 14 sera known to containhaemagglutinins. One serum gave the same titre(1 in 128) with both types of cell, but the othersagglutinated sheep cells to titres which werebetween two and 16 times higher than thoseobtained with human cells. Thus, not only werehuman cells less agglutinable than sheep cells, butthe discrepancies between the results were variableand could not be resolved by the use of a con-version factor. Similar discrepancies were notedwhen the tests were read by the deposit pattern.One serum from a non-tuberculous subject whichwas known to contain haemagglutinins was alsotested and gave identical titres (1 in 16) with bothhuman and sheep cells.

Sensitized human cells were valueless for thelysis test by the technique used, as lysis occurredwith only one of the 14 sera and in this case thelysis titre was 64 times lower than when sheep cellswere used. Ten of the 14 sera gave lysis of sen-sitized sheep cells at titres of 1 in 8 or over.

The Antigen.-Comparisons were made offour sensitizing antigens: dialysed old tuberculin(Burroughs Wellcome), dialysed old tuberculin(Evans), P.P.D. (Weybridge) 2.5 mg. per ml., andthe crude polysaccharide fraction of old tuber-culin described by Hilson and Elek (1951). Inthe comparative tests all serum dilutions were

made in bulk and then distributed into duplicateseries of tubes.

Six sera were tested by the haemagglutinationtechnique using sensitizing antigens prepared bydialysis of Burroughs Wellcome's and of Evans'old tuberculin. The titres in both cases were

found to be identica!.Hilson and Elek (1951) claimed greater speci-

ficity for an antigen derived from the partialfractionation of old tuberculin and described as

the crude carbohydrate fraction or C.C.F. This

66



j.com/

J Clin P


ownloaded from

http://jcp.bmj.com/


was prepared according to their directions, and acomparative series of tests on four sera from tuber-culous and five from non-tuberculous subjectswere put up by the haemagglutination and lysismethods, using cells sensitized with old tuberculinand with C.C.F. All these sera were known to con-tain haemagglutinins and four gave positive lysistests. The agglutination of cells sensitized withC.C.F. occurred more rapidly and was somewhatmore intense than with tuberculin-sensitized cells,but no significant difference was found betweenthe final titres for either the haemagglutination orthe lysis tests and the use of C.C.F. gave no evi-dence of increased specificity.

Purified protein derivative (Weybridge) was usedby Boyden (1951) as a sensitizing agent both fora modification of the Middlebrook-Dubos test andfor a test he described for the detection of anti-bodies to tuberculo-protein in which the cells werepreviously treated with tannic acid. Seventeensera were tested by the haemagglutination methodto compare the sensitizing effect of undiluteddialysed tuberculin with that of an equal volumeof 1 in 10 P.P.D., 2.5 mg. per ml. Identical titreswere obtained in all cases. Five further sera wereused to compare 1 in 10 P.P.D. with a 1 in 4dilution. Both concentrations were equally effec-tive. Purified protein derivative, 1 in 10, will sen-sitize sheep cells for the lysis test, although titreswere somewhat lower than when dialysed oldtuberculin was used. Higher concentrations ofP.P.D. were not tried.Experiments comparing the specific inhibition of

the haemagglutination reaction by P.P.D. and bya purified tuberculopolysaccharide indicated thatthe fraction of P.P.D. responsible for sensitizingthe cells was the 5% of contaminating poly-saccharide present in the preparation.

Sensitization of the Cells.-The effect of vary-ing three of the conditions of sensitization wasstudied.

Effect of Alteration of pH.-Dialysed tuberculinwas mixed with equal volumes of 0.1M phosphatebuffer solution and three batches of cells washed inunbuffered saline were sensitized at pH 6.4, 7.0,and 7.6. Haemagglutination titres and the degreeof agglutination were identical when these batcheswere tested with a known positive serum.

Effect of Diluting the Anztigeni.-Sheep cellswere sensitized with dialysed old tuberculin in con-centrations of 1 in 1, 1 in 2, and 1 in 4. Thesuspensions were then tested against a knownpositive serum. The degree of agglutination andthe end-titres were identical with the undilutedand 1 in 2 dilutions of the sensitizing antigen,

although agglutination was more apparent aftertwo hours at 370 C. with the undiluted material.Dilution of the tuberculin to 1 in 4 resulted in adiminution of titre of one tube and less markedagglutination. The antigen was therefore usedundiluted for all tests.

Effect of Variations in Temperature of Sensi-tization.-Cells treated with dialysed old tuber-culin for two hours at 370 C., 200 C., and40 C. were compared by haemagglutination witha known positive serum. Sensitization was morecomplete at 370 C. than at 200 C., and there wasno agglutination of cells treated at 40 C.

Effect of Variations in Size of Tube forHaenagglutination Test and in Quantities ofReagents.-Haemagglutination tests were per-formed in tubes of 12 mm., 9 mm., and 6 mm.diameter respectively, using varying volumes ofreagents. As in previous tests, the serum dilutionswere made in bulk and subsequently distributed tothe test racks in order to reduce any inherent tech-nical errors. There was no loss of sensitivity in using6 mm. tubes, and a total volume of 0.16 ml. wasfound to be most easily read for the haemagglutin-ation tests and gave identical titres to thoseobtained with 0.8 ml. amounts in the larger tubes.

Effect of Varying Period and Temperature ofthe Test-During the early stages of the investi-gation discrepancies were noticed between read-ings of the tests made after two hours at 370 C.and after standing overnight at room temperature.Some positive sera agglutinated to titre after twohours at 370 C., while others showed little or noagglutination at this time, but when left atroom temperature overnight agglutination becameapparent.

Thirteen sera from tuberculous and non-tuber-culous subjects containing haemagglutinins weretested against sensitized sheep cells at differenttemperatures for 16 hours. The tubes were wellshaken at two and four hours, as, in the testsincubated in the water-bath at 370 C., the cellssedimented more rapidly, and it was considereddesirable to keep all the cells in suspension for anapproximately comparable time. The results areshown in Table II.Of the seven sera from non-tuberculous cases

five gave higher titres at room temperature than at370 C., while the other two showed no change.Of the six sera from tuberculous cases twoshowed higher haemagglutinin titres at 370 C.than at room temperature, three gave identicaltitres, and one showed greater activity at roomtemperature. No absolute distinction between

67



j.com/

J Clin P


ownloaded from

http://jcp.bmj.com/

6. C. SHERRIS

TABLE IIEFFECT OF VARYING THE TEMPERATURE FOR

HAEMAGGLUTINATION TEST

Serum Titres

Serum 370 C Room 4 CNo. . Temperature 4'C-

_ 34 1/8 1/16 -

93 <1/2 1/4 -J92 1/2 1/16 -

Non-tuberculous 332 1/4 1/4 1/8sera 36 1/16 1/16 1/8

47 1/4 1/8 1/876 < 1/4 1/8 1/8

104 1/128 1/32 1/3281 1/128 1/64 -

Tuberculous sera 99 1/32 1/32 1/16186 1/64 1/64 1/6482 1/16 1/16 1/16103 1/16 1/32 1/16

haemagglutinins in tub2rculous and non-tuber-culous sera was detected by this method, but thesepreliminary observations suggest that there is atendency for them to have different optimumtemperatures of activity.

The Series and Methods of InvestigationThe main purpose of this section of the inves-

tigation was to compare the haemagglutination andlysis tests and to determine which of them, ifeither, would merit an extended investigation of itspossible use as a diagnostic test.The sera investigated comprised a selected

group drawn mainly from cases of active tuber-culosis and from presumably healthy subjects.The few sera from cases of other diseases andfrom doubtfully tuberculous patients were testedat a time when a clinical diagnosis of tuberculosiswas considered probable. Sera were obtainedfrom 150 subjects in all and their origin is shownin Table III.

TABLE IIIORIGIN OF SERA EXAMINED BY COMPARATIVE

HAEMAGGLUTINATION AND LYSIS TESTS

Category No. of Sera

Healthy blood donors 69Healthy antenatalpatients. 1 5Other healthy subjects. 11Patients suffering from diseases other than tuber-

culosis 3Cases of pulmonary tuberculosis.42Cases of pelvic tubercuosis .Cases of tuberculous meningitis 7? Tuberculous cases... 2

Total ! 150

Of the 11 sera taken from the group classifiedas " other healthy subjects," two had been inocu-lated with the vole bacillus six months previouslyand eight were tuberculin-negative. All but oneof these 11 cases had been previously tuberculin-

tested. Of the three patients suffering fromdiseases other than tuberculosis, one was bronchi-ectatic, one had a virus meningitis, and one was anapparently cured case of sarcoidosis whose tuber-culin reaction had recently converted. Two doubt-fully tuberculous cases were included in the series.In both of these the diagnosis was considered inview of a suspicious radiological opacity, but clinic-ally no evidence of active tuberculosis was found.These 150 sera were tested in groups of about

40, and haemagglutination and lysis tests put upsimultaneously, using the same batches of sensi-tized cells. The controls already described wereincluded for all sera, and a positive and negativeserum of known titre was put up with each batchof tests as a check on the sensitization of the cells.At the time of reading the tests the sera wereidentified only by a serial number, and it was notknown from which type of case they had origin-ated. Sera giving positive reactions throughoutthe dilutions of the test were subsequently re-tested at higher dilutions to determine the end-point.The results of tests on the 50 tuberculous and

98 non-tuberculous cases are shown as histogramsin Fig. 1.Most workers have considered titres of 1 in 8

or of 1 in 16 as positive for the haemagglutinationtest, and in this series a " positive " titre of 1 in 16or over gives the best separation of results. Forthe lysis test Middlebrook (1950) considered a titreof 1 in 8 or over as positive, and these results con-firm that this is the optimum point at which toplace an arbitrary diagnostic level.When comparisons between the two tests are

made with these titres as the dividing line betweenpositive and negative results, 12 of the 98 non-tuberculous cases give false positive results by thehaemagglutination method compared with six bythe lysis method, and 18 of the 50 tuberculouscases are " negative" by the haemagglutinationtest as against 16 by the lysis test. As the two-fold dilution techniques used in testing the serahave an inherent error of one tube, it is of impor-tance to compare the likelihood of such an errorchanging the interpretation of an individual test.Taking the "diagnostic " level at the titres indi-cated above, 49 of the results of the haemagglu-tination tests lie on either side of this arbitrarydividing line compared with only 20 of those ofthe lysis tests. The lysis test thus shows not onlya higher proportion of positive results in cases oftuberculosis and fewer false positives in non-tuber-culous cases, but also a greater separation ofresults on either side of the diagnostic level.

68



j.com/

J Clin P


ownloaded from

http://jcp.bmj.com/


Sera from Cases of Pul-monary Tuberculosis.-Serafrom 42 cases of pulmonarytuberculosis were tested.'Thirty of these were positiveby the lysis test and 29 bythe haemagglutination test.Three sera gave positivehaemagglutination but nega-tive lysis tests ; in one thehaemagglutination titre was1 in 128 and the lysis titre<1 in 4, a finding which was

confirmed in five samples ofserum from this patient re-

ceived over a period of threemonths. Four cases showedpositive lysis tests and nega-tive haemagglutination tests.One serum gave a haem-agglutination titre of 1 in 16only and a lysis titre of 1 in128, and it is of interest that a

positive Coombs reaction wasobtained on tuberculin-sensitized cells which hadbeen incubated for 30minutes at 370 C. with a 1

in 50 dilution of thisserum.

Information regarding theclinical condition of thepatients, of the Ministry ofHealth grade, and the resultof the last E.S.R. was sentwith each specimen. In some

TUBERCULOUS CASES

15

10

A

< 114 114 118 1116 1/32 1164 >1/64

TUBERCULOUS CASES B

80

70

60

NON-TUBERCULOUS CASES

IIIIIIII

NON-TUBERCULOUS CASES

50 1

40

30

20

10 I

< 114 114 118 1116 1132 1/64 >1164 < 114 114 1/8 1116 1/32 1164 >116FIG. 1.-A, results of the haemagglutination test on 148 sera; B. results of the lysis test

on 148 sera.

cases the information wasincomplete, but the clinician's opinion as to theclinical activity of the lesion at the time of takingthe specimen and the grade to which the patient'slesion was allotted are shown in Table IV andcompared with the results of the tests. The figures,though small, suggest a positive correlation betweenthe serological results and the activity of the

disease. A less subjective comparison is shownas a spot diagram in Fig. 2, in which E.S.R. resultsfrom cases of pulmonary or meningeal tuber-culosis are compared with the haemagglutinationor lysis titres of sera taken at approximately thesame time. These results indicate a well-markedcorrelation between the results of the E.S.R. and

TABLE IVA TABLE IVB

CORRELATION BETWEEN RESULTS OF HAEM- CORRELATION BETWEEN CLINICAL ACTIVITY OF

AGGLUTINATION AND LYSIS TESTS AND MINISTRY THE LESION IN 41 CASES OF PULMONARY TUBER-

OF HEALTH GRADE IN 40 CASES OF PULMONARY CULOSIS AND THE RESULTS OF THE SEROLOGICALTUBERCULOSIS TESTS

Haemagglutination LssTsMinistry of Health Test

Grade

Positive Negative Positive Negative

GradeA .. 2 2Grade BI.. 2 2 2 2Grade B2 .. 18 10 20 8Grade B3 .. .. 5 1 6

Haemagglutination Lysis TestClinical Activity Test

Positive Negative Positive Negative

Quiescent.. .. 2 2 2 2Regressing .. 12 6 12 6Stationary.. .. 6 0 5 1Progressing 10 3 12 1

69



j.com/

J Clin P


ownloaded from

http://jcp.bmj.com/

J. C. SHERRIS

> 1164

1164

1132

' 1/8E 8c)

I1/4

< 1/4

> 1164

1164

1/32

- 111116

1/8

114

0A

0

0 a

.3

s -

10 20 30ESR lmm/hr)

*

* 0 0

a0

a. UO.10 20 30

E S R Nmm/hr.FIG. 2.-A, correlation between haemagglutination

results; B, correlation betsreen Iysis titres and I

of the serological tests and particulysis test. Special mention should bepoint on this diagram referring tobetween 40 and 50 and a haemaggILof I in 4 and lysis titre of less thanpatient had a gross, superadded not

pulmonary infection and there was

doubt as to the activity of the tiiberAll but 10 of these cases had been

streptomycin or combined streptomycEight of these untreated cases gave pagglutination tests with titres of up toeight gave positive lysis tests with tin 128. This small group is a reascsentative sample of the whole series.

Sera from Cases Presenting ITuberculous Meningitis.-The sera

cases of tuberculous meningitis were

were positive by both tests and four n

of those giving positive results w

active and were suffering from pulrrculosis in addition to the meningealboth patients died. The other patiena positive result was obtained was a

complicated tuberculous meningit;s N

was clinically considered to be a

E.S.R., however, was 59 mm. in orwas subsequently discharged as cured

patients giving negative serological tests the diseasewas definitely arrested in three and probably inthe fourth: one of these patients had previouisly

* suffered from pelvic tuberculosis which was treatedsurgically, and one had an inactive pulmonary

* lesion treated by artificial pneumothorax. Thexrwere all subsequently discharged as cured.

Sera from Blood Donors.-Sixty-nine specimens* of sera were received from healthy blood donors.

Sixty of these were negative when tested by the40 50t haemagglutination method and 65 were negative

by the lysis test. Six sera gave a positive haem-agglutination test and negative lysis test in fourof these the lysis titre was less than 1 in 4, andin the other two lysis was apparent to a titreof I in 4 only. One serum gave haemagglutination

* to a titre of I in 8 (negative) and lysis to a titreof I in 16.Three of these sera were thus positive by both

the haemagglutination and lysis tests, and inquiriesas to the clinical condition of these subjectsrevealed no evidence to suggest that they weretuberculous, but it was not possible to subject

40 50+ them to a full physical and radiological examin-ation.

titres and E.S.R.E.S.R. results. Sera from Antenatal Patients. Fifteen seralary1of the from healthy antenatal patients were tested.ardy of the Fourteen of these were negative by both themade of the haemagglutination and lysis tests and one wasan E:S.R of positive by both methods: no evidence of tuber-itinationtltre culosis or other illness was found in this case at1 in 4. This routine antenatal examination, but it has notn-tuberculous been possible to re-examine her physically oiconsiderableculous lesion.treated with Sera from Other Non-tuberculous Healthv

in and P.A.S. Subjects.-Sera were tested from 11 other healthxositive haem- non-tuberculous subjects. All but one of these

1 in 32, and had previously been tuberculin-tested and two had:itres up to 1 been inoculated with the vole bacillus vaccine eight)nably repre- months previously. Two of the 11 sera gave

positive hae;nagglutination tests, and all were

Primarily as negative by the lysis test. The relevant data on thefrom seven 10 patients who had been tuberculin-tested is

tested. Three summarized in Table V. These results give no

egative. Two direct support to the view that tuberculin-testingere clinically per .se may give rise to haemagglutinins or lysins toionary tuber- tuberculin-sensitized cells. Further evidence on

lesions, and this point is presented below.t from whom Sera from Cases of Non-tuberculous Disease.-A case of un- Sera were tested from three patients only. Onewhose disease was suffering from mumps encephalitis and gaveLrrested: her negative results by both the haemagglutinationne hour. She and lysis tests: one had a collapsed and bronchiec-Of the four tatic left lung and similarly gave negative sero-

70

< 114



j.com/

J Clin P


ownloaded from

http://jcp.bmj.com/


logical results. The third was a case of apparentlyhealed pulmonary sarcoidosis. This patient was ingood health, and had a normal E.S.R. and negativeradiograph, but the Mantoux test had converted topositive within the previous six months. Thehaemagglutination test was negative but the lysistest positive in this case (HA. 1/8, L. 1/16). Thissubject was a laboratory worker and exposed toinfection by tubercle bacilli.

Sera from Cases of Suspected Tuberculosis.-Sera were tested from two cases with suspiciousradiological lesions. Both were clinically in goodhealth and have remained so. One gave a positivehaemagglutination and negative lysis test (HA.1/16, L <1 /4) and the other was negative by bothtests (HA. I /4, L. <1/4).

Effect on the Haemagglutination and Lysis Testsof Previous Tuberculi Injections

The results of tests on the sera of 10 normalsubjects are sum.marized in Table V and have

TABLE VSEROLOGICAL FINDINGS ON 10 SERA FROMSUBJECTS PREVIOUSLY TUBERCULIN-TESTED

Previous Mnh irSerum Vole since Last Mantoux HamaguNo. Tuberculin Result Hamsl-LysisVaccineTest tinmation TestVcine es Test

55 No 5 1+ve 1'8 < 1 456 Yes 5 -ve 1'16 <1 457 . 8 j+ve 4 < 1 4105 No I I -ve < 14 < 14106 . I -ve <<1'4 <1 4107 .. I -e 1/16 < 1 4108 ,, I -ve 1 4 < 1 4114 ,, I -ve 18 <14115 -I yve <1 4 <1 4116 ,. -ve 1 4 <1 4

already been referred to. When these results are

compared with those in Fig. 1 for all non-tuber-culous subjects they may be seen to be a represen-

tative sample. Serial tests were also made on

the sera of three non-tuberculous, tuberculin-positive subjects following the intrathecal inocula-tion of 0.00375 mg. of P.P.D. The sera were takenboth before the inoculation and one and five daysafterwards; in one-case a further test was madeseven days after the injection. No rise of haem-agglutinin or lysin titres occurred in these cases.

More significant evidence of the failure oftuberculin inoculations to cause the developmentof lysins was obtained by the repeated examinationof sera from patients under treatment with strepto-mycin and intramuscular tuberculin inoculations.Six such cases were studied, and all the specimens

of sera from each case were tested together understandard conditions.

F.B., aged 45, is cited as an example. Nine serawere taken over a period of two months duringwhich a full course of tuberculin and streptomycintreatment was given. This patient had extensivefibrosis, bronchiectasis, and emphysema, and it wassubsequently believed that the principal factor inhis infection was non-tuberculous. The results aresummarized below:

Date Tuberculin Dose Haemagglutina- Lysis(ml.) tion Titre Titre

27.12.51 0-002 1,4 < 1 415.1.52 006 1/8 1 428.1.52 0 1 1/4 <142.2.52 _ < 1/4 <1 47.2.52 0-2 <1/4 <1415.2.52 04 <1 4 <1421.2.52 0-6 14 <1 422.2.52 1-4 <1 427.2.52 1'8 <1 4

These results, together with those from theother five patients, indicate that some slight rise inhaemagglutinin titre may follow the intramuscularinoculation of high doses of tuberculin; this is,however, an irregular effect. The lysis titre is notsignificantly affected by injections of large dosesof tuberculin.

DiscussionNumerous serological tests for antibodies to

M. tuberculosis have been described (Wilson andMiles, 1946), but none has yet been generallyaccepted as a practical advance in the diagnosisof the disease. The haemagglutination test ofMiddlebrook and Dubos (1948) promised to givebetter results than those used previously, andGernez-Rieux and Tacquet (1950b) in a compar-ative series demonstrated the superiority of thehaemagglutination test over complement-fixationtests, using both the Besredka egg antigen and theN'egre and Boquet antigen. In almost all reportedseries of haemagglutination tests, however, therehave been a proportion of false positive results,and the findings described in this paper are verysimilar to those obtained with similar techniquesby Fleming et al. (1951), Kirby et al. (1951), andMollov and Kott (1952), in which haemagglutininswere detected in the sera of about half the non-tuberculous subjects investigated and agglutinationoccurred to "diagnostic " titre in about 10% ofthese cases.

It is clear that if the haemagglutination test isto be of practical diagnostic value some meansmust be found to increase its sp2cificity. The useof human Group 0 cells decreases the sensitivity

71



j.com/

J Clin P


ownloaded from

http://jcp.bmj.com/

J. C. SHERRIS

of the test markedly and in an irregular fashion,and although it eliminates the necessity of pre-

liminary absorption of the sera the results are lesssatisfactory than with sheep cells: variation ofthe period and temperature of incubation alsoseems to offer little prospect of any considerableimprovement of the test. Some workers haveclaimed greater specificity with the use of partiallypurified fractions of tuberculin, and although no

difference in the agglutination titres was found incomparative experiments with four different anti-gens in -this investigation, it seems possible fromthe work of Pound (1952) that further fraction-ation of the polysaccharide antigens may improvethe results.The lytic modification of the test shows greater

promise as a useful method for the diagnosis oftuberculosis, and the comparative series of the twotests reported here confirm the findings of Middle-brook (1950) and Mollov and Kott (1952) that itis more specific for the disease. The discrepanciesbetween the haemagglutination and lysis titres on

individual sera also support the view of Middle-brook (1950) that a different antibody is responsiblefor the reaction, and the lysis test certainly meritsfurther study in view of its superiority to thehaemagglutination method.

In any future assessment of the haemolyticmodification the method should include a com-

plement titration and a more accurate dilutiontechnique covering a closer range of dilutions thanthose used here. One such method has been de-scribed (Maillard and Gagliardo, 1951 ; Maillard,1952), and has apparently proved satisfactory inpractice.Some investigations have been reported of the

relationship between haemagglutinin titres and theextent, severity, and prognosis of the tuberculousdisease (Gernez-Rieux and Tacquet, 1950b ; Sohier,Juillard, and Trimberger, 1950; Spain, Childress,and Rowe, 1952; Hinson et al., 1952), but muchless work has been done on this aspect of the sub-ject with the lytic modification of the test. Theresults of this series suggest that the lysis titre isrelated, in part at least, to the activity of the lesion,but the cases were not followed up for long enoughto show what other factors may be involved. It willbe of great interest to determine whether these anti-bodies are related in any way to the development ofimmunity in tuberculosis; but any such study inhuman cases presents considerable difficulties, as

the response to the infection is almost invariablymodified by the therapeutic measures employed.The development of antibodies to tuberculin-

sensitized cells following intradermal tuberculin

inoculations was described by Smith and Scott(1950) and by Gerstl, Kirsh, Andros, Winter, andKidder (1952) and would, if confirmed, have to betaken into account in the interpretation of the test.In the cases reported here, however, neither largeintramuscular doses of tuberculin nor previousMantoux testing affected the lysis test, although asmall rise in the haemagglutinin titres was some-times detected. There is thus no evidence thatprevious tuberculin inoculations will give rise tofalse positive lysis tests if the technique describedhere is used.

In conclusion, the results of this investigationshow that the test for lysis of tuberculin-sensitizedsheep cells in the presence of antibody and com-plement is more specific for tuberculosis than thehaemagglutination test, and it is considered thatfurther extended studies of this method should becarried out to assess its value as a diagnostic andprognostic test. It will be important, however, inany such investigation to be able to subject healthyand other presumably non-tuberculous subjects tofull radiological and c!inical investigation if neces-sary in addition to studying repeated samples ofserum from tuberculous patients throughout thecourse of their disease.

SummaryThe Middlebrook-Dubos haemagglutination test

for tuberculosis was compared with its lytic modi-fication; the lysis method was shown to be morespecific as a diagnostic test.Marked discrepancies between the two tests

performed on individual sera were found andevidence was presented to support the postulatethat different antibodies are concerned.Experiments were undertaken to determine the

effect of varying some of the conditions and re-agents for the haemagglutination test. Sheep cellswere shown to be superior to human cells, andalterations in the duration and temperature ofincubation caused wide variations of the finaltitre. Human cells were found to be useless for thelysis test by the technique described.

Four sensitizing antigens were compared forthe haemagglutination test and no significantdifference was detected between them.

Massive doses of intramuscular tuberculin wereshown to have little effect on the results of thehaemagglutination test and none on the lyticmodification.The haemagglutination test described was con-

sidered to be of little value in the diagnosis oftuberculosis, but the lytic modification should befurther investigated.

72



j.com/

J Clin P


ownloaded from

http://jcp.bmj.com/


I wish to thank the many clinicians who suppliedsera and details of their cases, especially Dr. L. P. A.Newborne, Dr. W. S. Hamilton, and Dr. Jean M.Grant. My thanks are also due to Dr. R. L. Vollumfor his constant advice and encouragement, to Dr.Arthur Spriggs for correcting the manuscript, and toMrs. D. J. Trapnell and Miss Beryl Hall for theirhelp in preparing the typescript.

REFERENCESAdcock, J., Haley, R. R., and Davey, W. N. (1951). J. Lab. clin.

Med., 38, 736.Boyden, S. V. (1951). J. exp. Med., 93, 107.Eagle, H. (1937). The Laboratory Diagnosis of Syphilis. St. Louis.Fisher, S., and Keogh, E. V. (1950). Nature, Lond., 165, 248.Fleming, J. W., Runyon, E. H., and Cummings, M. M. (1951).

Amer. J. Med., 10, 704.Gerstl, B., Kirsh, D., Andros, E. M., Winter, J. W., and Kidder,

L. E. (1952). Amer. J. clin. Path., 22, 337.Gernez-Rieux, C., and Tacquet, A. (1949). Bull. Acad. Med., Paris,

133, 556.- (1950a). Ann. Inst. Pasteur, 78, 550.

-- (1950b). Ann. Inst. Pasteur Lille, 3, 1.

Hilson, G. R. F., and Elek, S. D. (I195 1). Journal ofClinical Pathology,4, 158.

Hinson, K. F. W., Jones, A. Richardson, and Chamberlin, J. A.(1952). Brit. J. Tuberc., 46, 50.

Kirby, W. M. M., Burnell, J. M., and O'Leary, B. (1951). Amer.Rev. Tuberc., 64, 71.

Maillard, E. R. (1952). Amer. J. publ. Hlth, 42, 175.and Gagliardo, F. J. (1951). Amer. Rev. Tuberc., 64, 675.

Middlebrook, G. (1950). J. clin. Invest., 29, 1480.and Dubos, R. J. (1948). J. exp. Med., 88, 521.

Mollison, P. L. (1951). Blood Transfusion in Clinical Medicine, p. 18.Oxford.

Mollov, M., and Kott, T. J. (1952). Amer. Rev. Tuberc., 65, 194.Pound, A. W. (1952). J. Path. Bact., 64, 131.Rothbard, S., Dooneief, A. S., and Hite, K. E. (1950). Proc. Soc.

exp. Biol., N. Y., 74, 72.Scott, N. B., and Smith, D. T. (1950). J. Lab. clin. Med., 35, 303.Sievers, O., Ulstrup, J., and Winblad, S. (1950). Nord. Med., 43, 667.Smith, D. T., and Scott, N. B. (1950). Amer. Rev. Tuberc., 62, 121.Sohier, R., Juillard, J., and Trimberger, I. (1950). Ann. Inst.

Pasteur, 78, 283.-Trimberger, I., and Juillard, J. (1950). Ibid., 79, 347.Spain, D. M., Childress, W. G.,and Rowe, C. (1952). Amer. J. clin.

Path., 22, 86.Thalhimer, W., and Rowe, C. (1951). Amer. Rev. Tuberc., 63, 667.Thomas, J. C., and Mennie, A. T. (1950). Lancet, 2, 745.Wilson, G. S., and Miles, A. A. (1946). Ed., Topley and Wilson's

Principles ofBacteriology and Immunity, 3rd ed. London.

73



j.com/

J Clin P


ownloaded from

http://jcp.bmj.com/

MIDDLEBROOK-DUBOS HAEMAGGLUTINATION …J. clin. Path,. (1953), 6, 64. THE MIDDLEBROOK-DUBOS...

Documents

Transcript of MIDDLEBROOK-DUBOS HAEMAGGLUTINATION …J. clin. Path,. (1953), 6, 64. THE MIDDLEBROOK-DUBOS...