Studies on a Serum with Peculiar Haemagglutinating Properties

Vox Sang. 6: 409-428 (1961)

Studies on a Serum with Peculiar Haemagglutinating Properties

MORTEN HARBOE, TROND REINSKOU, HELCE HEIsTii and PAL BJORNSTAD

Institutes for Thrombosis Research and Forensic Medicine, University Hospital (Rikshospitalet), Blood Bank, Ullevll Hospital, and Central Laboratory,

Aker Hospital, Oslo, Norway

During compatibility tests prior t o blood transfusion, it was ob- served that red cells which had been exposed t o serum K.G. were agglutinated when subsequently washed in saline. Studies were made t o elucidate the serological and biochemical basis for this peculiar phenomenon. The present paper describes these investigations which showed that the responsible substance was a y-globulin of high molecular weight, also possessing some but not all of the usual rheu- matoid factor activities.

Case Report

The patient was a man born in 1893. He had been in good health until 1952, when he suffered from an acute myocardial infarction. Anticoagulant therapy was maintained until 1958. Except for some anginal pain, he enjoyed good health during these years. He had for some years occasional lumbar pains, but no clinical signs of rheumatoid disease.

I n April 1959, he began to suffer from anorexia, slight fever, and he felt exhausted. Occasionally, he noticed darkcoloured urine. He was admitted to Medical depart- ment B, Aker Hospital, in May 1959, and was readmitted several times during 1959 and 1960. The physical and clinical findings were practically unchanged during the whole period. Except for slight oedema, physical findings were normal. The urine contained protein, occasional white and red cells, hyaline and granular casts. Blood group A, MN Rh 4- (probable genotype R,R,). WR negative. He had a moderate anaemia, haemoglobin varying between 11.0 and 11.8 g./100 ml., with a red cell count of 3.6-3.9 mill./mm3. Leukocytes : normal total and differential counts. Thrombocytes 352,000 per mm3. Peripheral blood smears showed no definite abnor- malities. Sternal marrow smears showed slightly increased erythropoiesis. Sedimen- tation rate 70-75 mm. in one hour. Reticulocytes: 26-60°/,,. Serumiron 180 y/100 ml. Direct Coombs’ test (citrate milien, see below) negative. Indirect Donath-Landstei- ner test: negative. Tests for warm and cold haemolysins: negative. Determination of the life span of the patient’s own Crsl-labelled red cells showed a reduced survival time (T/2: 16 days). Paper electrophoresis of serum proteins showed a marked

29 Vox Sang., Vol. 6 , No. 4 (1961)

410 H a r b o e , R e i n s k o u , H e i s t o , H j o r n s t a d

decrease of the albumin fraction (2.2 g./100 ml.), and an increased y-globulin frac- tion (2.4 g./100 ml.). Starch gel electrophoresis showed that haptoglobins were present in normal or slightly increased amounts. Liver function tests: Icteric index 4 (Meulengracht); Thymol turbidity test 20 Maclagan units; Alkaline phosphatases 2.3 Bodansky units; Prothrombin-Proconvertiri 50 per cent, with no increase after intravenous vitamin K administration. There were signs of impairment of renal function with a serum creatinine content of 2.6 mg./100 ml.

A tentative diagnosis of chronic pyelonephritis and cirrhosis of the liver was made. The anaemia was largely unexplained, but a renal factor was probably and a slight haemolytic component definitely present.

Because of the anaemia, the patient received blood transfusions, totalling 4 liters of blood from October 1959 to January 1960. Compatibility tests in saline were made prior to these transfusions with a technique which would not permit the observation of the phenomenon to be described. A new transfusion was planned in February 1960. Prior to this transfusion, compatibility tests were attempted by the indirect Coombs' technique. It was then observed that red cells which had been exposed to the patient's serum were agglutinated when subsequently washed in saline. Further transfusions were therefore not given.

On March 20th, the patient fell abruptly ill and died in a few minutes with the clinical picture of acute cardiac failure. Post-mortem examination revealed general arteriosclerosis, cardiac hypertrophy with fibrous scar tissue suggesting previous myocardial infarction, marked congestion and pulmonary oedema. Microscopic examination of the liver showed marked congestion with some fibrous tissue pro- liferation. The kidneys showed the typical picture of chronic pyelonephritis.

Materials

Pooled human y-globulin was a gift from AB Kabi, Stockholm, Sweden. The preparation was from batch no. 70,347 of their commercial preparation. Solid ;.-globulin was prepared by heating a t 75" C. for 3 minutes [22].

Human albumin was the commercial preparation of AB Kabi, batch no. 67,103. Red cells were obtained from the blood bank of the University Hospital. The cells

were stored for up to 4 days before they were used. All sera were stored in small aliquots a t -2.5" C.

Agammaglobulinaemic serum. The sample contaiued less than 25 mg. y-glo- bulin/100 ml., as determined by immune precipitation. The determination was kindly performed by Dr. H. Fudenberg of The Rockefeller Institute, New York, N.Y.

Rabbit anti-human serum (K 12.5) was used for immunoelectrophoretic iuvesti- gations. The rabbit had been immunized by the iutraverious route with pooled human serum.

Coombs' serum K 310 had been prepared by similar immunization of a rabbit. I t contained both anti--;-globulin and anti-Tion-y-globuliri antibodies, as evideuced by the controls shown in Table VI.

Rabbit serum (R 46.5) containing antibodies against no other human serum proteins than 7 s and 1 9 s y-globulins was kindly provided by Dr. H. Fudenberg. Controls for activity are shown in Table VI.

Rabbit amboceptor active against human red cells was a gift from Dr. L. Podlia- chouk of Iristitut Pasteur, Paris, France.

Studies on a Serum with Peculiar Haemagglutinating Properties 411

Incomplete ant i -Rh sera Mu. and Ri. were used for coating of red cells for demon- stration of rheumatoid factor activity. Their properties have been described before [14].

Ethanethiol (ethyl mercaptan) was The British Drug Houses' preparation (British Drug Houses, Poole, England).

Akryl-plast particles were kindly given by AB Bofors Nobelkrut, Bofors, Sweden.

Methods

Agglutination tests were made with standard techniques. Unless otherwise indicated, the recorded dilution indicates the original dilution of serum when introduced into the tubes. For agglutination tests in tubes, an equal volume (0.2 ml.) of serum dilution and a 2 per cent suspension of red cells were mixed. The tubes were allowed to stand for four hours, and the red cell sediment removed for micro- scopic examination. In most tests using serum K.G., the cells were spun down before reading. Tests with red cells coated with incomplete anti-D were made on slides as previously described [12, 131. Inhibition experiments were made using one volume of serum diluted to contain about 10 hemagglutinating doses, one volume of the material to be tested for inhibiting capacity and one volume of a 2 per cent suspension test cells. Proper controls were included as for studies on the Gm system

Testsfor rheumatoidfactor activity. The tests were made incitratemilieu to prevent agglutination of untreated cells (see below). Controls with known positive and negative sera to ascertain that citrate did not change the reactions were always in- cluded. The Waaler-Rose test was made with human group 0, rr red cells [27] to avoid absorption of heterophile agglutinins. The sensitizing amboceptor was used in !/1 of minimum agglutinating dose. F 11-akryl particle tests were made according to Winblad [38, and personal communication] with slight modifications : The suspension was stabilized with human albumin, and 0.15 M Sigma buffer pH 8.2 was used. The results were read macroscopically without centrifugation after sedimentation over- night a t 37' C. Precipitin tests were made in capillary tubes, and the interphase precipitate between rheumatoid factor and y-globulin previously heated at 63" C. for 10 minutes visualized.

Absorption with solid y-globulin. One volume of serum diluted 1 : 10 was absorbed for one hour a t 37" C. with solid y-globulin prepared from an equal volume of a 2 per cent solution of pooled human y-globulin. The y-globulin precipitate was washed three times in saline, and great care was taken to suspend the final packed precipitate in a finely divided state in the serum to be absorbed. When attempts were made to absorb low-titered activities, minor dilutions of serum were used, the amount of solid y-globulin being increased correspondingly. For immunoelectro- phoretic investigations, undiluted serum was absorbed.

Treatment with ethanethiol was made according to Grubb and Swahn [l I] , exposing one volume of serum K.G. diluted 1 : 5 to one volume of 0.2 M ethanethiol for 20 hours a t room temperature. The different rheumatoid factor activities of two control rheumatoid sera decreased markedly or disappeared completely by similar treatment.

Analytical ultracentrifugation was kindly performed by R . Djurtoft of The Carls- berg Laboratories, Copenhagen, Denmark.

~ 3 1 .

412 H a r b o e , R e i n s k o u , H e i s t o , B j o r n s t a d

Immunorlertrophor~sis was performed according to the micro-method of Srhei- degger [28] with some minor modifications described by Clausen [3].

Anion eurhange rhromatography w as performed according to Sober, Gutter, Wyrkofland Peterson [29]. A stepwise elution technique [ l] was used to get maximal separation of 7s and 19s -:-globulins. Two ml. serum were put onto a column of t w o g. \.A-diethylaminoethylcellulose “ Eastman” (DEAE-cellulose), and the proteins Here eluted in fibe ml. portions. The total volume of eluting buffers was 150 ml. The protein content of each portion was measured by determining optical density a t 280 my. The portions representing each peak of eluted protein were mixed together, concentrated by ultrafiltration down to the original serum volume. made isotonic l)y dialjsis against 0.9 per cent saline and tested for agglutinating capacity. The protcin content of each fraction was qualitatively controlled by immune electro- phoresis.

Experimpnts and Results

PrP1irninar.y Observations

Red cells were incubated with serum K.G. When subsequent at- tvmpts were made to wash the cells in saline, strong agglutination was observed. The same phenomenon was observed with the patient’s own rrd cclls, and with cells from a number of donors of diffcrent blood groups not including groups B and AB. Washing experiments were then made with the following solutions: ACD solution, 3.1 prr cent trisodium citrate dihydrate, Ringer’s solution, 2 per cent EDTA in saline with pH adjusted to 6.5, heparin in saline (5 units per ml.), 0 prr cent Dextran in saline, 4 per cent human albumin, 30 per crnt bovine albumin and 1 per cent human y-globulin. Strong agglutina- tion was observed in all instances except when ACD, citrate or human y-globulin were used for washing. No significant difference in haemag- glutinating capacity of serum K.G. was found between pH 6.0 and 9.0. When previously washed red cells were incubated with swum K.G. diluted in citrate, no agglutination of the cells was observed if thc cells sedimented spontaneously, or after slight centrifugation. If the centrifugation was hard, some agglutination was observed. The prevention of agglutination by citrate was therefore judged to be relative and not absolute.

Group 0, Kh- ( r r ) red cells previously washed three times in saline w e n * then set to react with serial twofold dilutions of serum K.G. in saline. The results arc shown in Table I.

The red cells were strongly agglutinated by serum K.G., and a pronounced prozone phenomenon was observed. If, after the in- cubation period, the tubes were filled up with saline and the cells

Studies on a Serum with Peculiar Haemagglutinating Properties

TABLE I

Agglutination of Washed Red Cells by Serum K.G.

413

Group 0, Rh -(rr) red cells were washed three times in saline and incubated with serial twofold dilutions of serum K.G. in saline for ten minutes. (a) The mixture was centrifuged, and the sediment removed for microscopic examination. (b) The tubes were filled up with saline before centrifugation, and the sedimented cells were

examined for agglutination.

Reciprocal of dilutions 2 4 8 16 32 64 128 256 512 1,024

- + t - + + + + - t + ~ t + I - t - (a) -

(b) ++ ~ k + + ++ + -t + -t t t + +++ + t + ++ f -

spun down before reading, no prozone phenomenon was present and the agglutinating titer increased somewhat. The prozone phenomenon was observed with all red cells tested, including the patient's own cells. The agglutination was equally strong at 4", 20" or 37" C., and equally active with red cells from adults of groups 0, A, and A,, and with cord blood cells (six samples of groups 0 and A).

The agglutinating activity of serum K.G. against enzyme treated red cells was compared with untreated cells washed in saline. Cells

TABLE I1

Absorption of Serum K.G. with Washed Red Cells One volume of undiluted serum K.G. was absorbed once with four volumes of previously washed, packed red cells of different ABO groups for 20 minutes a t 20' C. The supernatants were tested for agglutinating capacity against group 0 red cells previously washed four times in saline. For comparison, serum K.G. diluted 1:20 was absorbed with an equal volume of packed washed red cells of group 0. (Four times absorption of undiluted serum with washed red cells resulted in slight reduction in titer which might be ascribed to inevitable dilution of serum

during absorption; not included in the table.)

Dilution of ABO group of Agglutination strength, reciprocals of dilutions serum K.G. cells used

for absorption 40 80 160 320 640 1,280 2,560

undiluted A, -++ ' 1 t 4 A t t ( I ) -

undiluted B 'A* + t + i i - - - -

undiluted 0 t + ' t + + + tS L t I ' -

1:20 0 - - - ~

Control -++ + t - t t ' I I 1 ' -

~ -

414 H a r b o e , R e i n s k o u , H e i s t o , B j o r n s t a d

treated with trypsin were weakly agglutinated and cells tested with papain and ficin were not agglutinated by any dilution of serum K.G.

Attempts were made to absorb the haemagglutinating substance with washed group 0 and A, red cells. When undiluted serum was treated with these cells, no reduction in agglutinating activity was observed. When the serum had been diluted 1:16 or more before absorption, all activity was removed as shown in Table 11. The absorption with group B red cells is discussed later.

The agglutinating activity was unchanged or only slightly reduced after heat inactivation (30 minutes a t 56" C.). After heating at 63" C. for 15 minutes, agglutinating activity disappeared.

Immunoelectrophoretic and Chromatographic Investigations

Immune electrophoresis of serum K.G. showed a distinct line in the P,-M region. The line was much stronger than in normal control sera where this line was barely visible, and of about the same strength as the P,-M line in a rheumatoid serum (H.H.) with strong rheumatoid factor activity. The P,-M line became definitely weaker in serum K.G. after absorption with solid y-globulin, but it did not disappear.

The antiserum was absorbed with an equal volume of human group 0, rr red cells which had been agglutinated by serum K.G. (The antiserum had been stored for some years in the deepfreezer and had no haemolyzing capacity towards human red cells.) Prior to this absorption the agglutinates had been thoroughly washed in saline. When the immune electrophoretic pattern of serum K.G. and the control rheumatoid serum was developed with this absorbed anti- serum, the P,-M line in both sera had disappeared completely, whereas no significant changes could be observed in any other lines. The immune electrophoretic patterns are shown in Fig. 1.

Fig. 2 shows the results of chromatographic fractionation of serum K.G. on DEAE-cellulose. In the four fractions obtained, agglutinating activity was found only in fraction 4, where it appeared without the prozone phenomenon observed in whole serum. Immune electro- phoretic control of the four fractions showed that fraction 1 consisted of pure y-globulin of low molecular type (7s y-globulins), fraction 2 contained P-globulins, fraction 3 the main part of the albumin together with wglobulins, and fraction 4 contained all demonstrable P,-M-globulin together with the rest of albumin and q lobul ins .

Studies on a Serum with Peculiar Haemagglutinatirlg Properties 415

Serum K G ,p2M

Norm a1 controls

1

Rheumatoid serum H. H.

K G. absorbed wi th solid 8glob

K. G. w i th absorbed antiserum

Rheumatoid serum H H w i th absorbed antiserum

0

I Fig. 1. Immunoelectrophoresis of serum K.G. and different controls (see text).

Only the immune globulins are shown in the Figure.

Tests for anti-B activity in citrate milieu also showed all activity confined to the 4th fraction.

Dissociation Experiments with Ethanethiol

The immune electrophoretic and chromatographic investigations indicated that the haemagglutinating activity was due to a macro- globulin of the P,-M class. Different serological activities due to such proteins are known to disappear by treatment with sulfhydryl com- pounds [7, 11, 22, 231. By treatment with ethanethiol, the ability of serum K.G. to agglutinate washed red cells disappeared, and the tests for rheumatoid factor activity (see below) became negative.

410 Harboe , Ke inskou . Heis t i i . Bjorristatl

5 1 2 i c

? 321

n L ...

30 60 110 150 mi elut ing f i u / d

+pH63+59,58---+-5 2 - d rnoi 0 0775 004 0 1 0 4

Fig. 3. Chrornatography of serum K.G. 0 1 1 DEAE-rellulose rolumtl.

Ultracentrifugal Analysis

Analytical ultracentrifugation of whole serum K.G. demonstrated the presence of a macroglobulin with a sedimentation coefficient of approximately 20S, amounting to 3.8 per cent of the total protein content. Ultracentrifugal patterns of serum K.G. are shown in Fig. 3.

Fig. 3. Analytical ultracentrifugation of serum K.G. Sedimentation is from left to right. The photographs were taken at 10 arid 30 minutes after obtaining full speed (1,000 REV/Sec). The macroglobulins with sedimentation constant s = 20s are

represented by the peek which is seen to move from left to right.

Studies on a Serum with Peculiar Haemagglutinating Properties 417

Further Characterization of the Haemagglutinating Substance

A wide variety of substances with different serological activities belongs to the B,-M class of proteins. Among them are Waldenstrom macroglobulins, rheumatoid factors, cold haemagglutinins, heterophile haemagglutinins both naturally occurring and in infectious mono- nucleosis, isohaemagglutinins anti-A and anti-B and saline agglutinat- ing anti-Rh [23]. Attempts were made to show whether serum K.G. showed some of these activities.

TABLE I11

Tests for Rheumatoid Factor Activities

The tests were made in citrate milieu to prevent agglutination of uncoated cells, controls with such cells were consistently negative. FII-akryl particle test was run

in the absence of citrate. Reciprocals of titers are given.

Test Normal sera Serum K.G. ~ ~~

Modified Wader-Rose <32 2 FII-akryl particle test < 20 320 Anti-D Mu < 8 640 Anti-D Ri <32 1,280

- y-globulin precipitation ++

The results of tests for rheumatoid factor activities are shown in Table 111. The Waaler-Rose test was completely negative. Some of the tests where human y-globulin is used as reactant were however strongly positive. The rheumatoid factor is known to precipitate directly with y-globulin (Epstein, Johnson and Ragan [S]) and may be absorbed on to solid y-globulin prepared by heat precipitation. The latter phenomenon is regarded so specific for rheumatoid factor that it has formed the basis for its isolation from serum [22]. Table IV shows the results of absorption experiments with solid y-globulin and serum K.G. together with control sera with different serological activities due to P,-M proteins.

The experiments confirmed that this absorption procedure is nearly specific for rheumatoid factor. Isohaemagglutinins, cold haemag- glutinins, heterophile agglutinins and saline agglutinating anti-Rh antibodies were not significantly reduced in activity. In contrast, tests for rheumatoid factor activity in which human y-globulin is

50 Vor Snag.. Vol. 6 . No. 4 (1961)

418 I1 ar l ) o r , Reins k ou, H c i i t 6 , U j orn s tad

TABLE IV

Absorption with Solid y-globulin

All sera, except the rheumatoid sera and serum K.G. were tested for one activity only. For technique of absorption, see text. Reciprocals of titers are given, numbers

i n italics denote significant reduction by the absorption.

Activity No absorption Absorption (controls)

1. Isohaemagglutinins A-sera (anti-B) 0-sera (anti-A) 0-sera (anti-B)

2. Cold haemagglutinins

3. Heterophile haemagglutinins (Paul-Bunnel test)

4. Saline agglut. anti-Rh (anti-CD, -D. -D)

5. Rheumatoid sera F 11-akryl particle test Waaler-Rose test Anti-D Ri. Isohaemagglutinins

anti- A anti-B

Heterophile agglutinins

6. Serum K.G. FII-akryl particle test Anti-D Ri. Anti-D Mu. Agglutination of washed red cells Anti-B

160,320,160,320,320,640 1280, 320, 320, 160 640, 320, 80, 40 640, 10,000, 640, 80, 80

320, 640, 320, 80

768, 640, 320

1280, 2560, 256, 80 640, 640, 128, 160 1280, 320, 256, 80

_ _ _ , , , 4 0 160, 160, 128, 40 20, 10, 10, 10

320 1280 640

640 8

160,160,160,320,160,640 1280, 320, 160, 80 640, 320, 40, 40 640, 5000, 640, 80, 80

160, 640, 160, 80

768, 640, 320

_ _ _ 9 7 , 4 0

160, 160, 4, 20 10, 10, 10, 10

<10 <10 ( 1 0

no agglutination < 2

used as reactant (FII-akryl particle test, red cells coated with ‘‘ diagnostic ” anti-D), became markedly reduced by the absorption procedure. The titer reduction in the Waaler-Rose test varied from serum to serum; in some instances the reduction was marked, whereas other sera had to be absorbed several times to get a marked reduction in titer. The heterophile agglutinin titer of the rheumatoid sera tested did not change by the absorption procedure. I n one serum, the titer of anti-B was markedly reduced during absorption.

Stiidiei 011 a Seriml with Peculiar Haemagglutiiiating Propertici 419

After absorption with solid y-globulin, serum 1C.G. lost its abilit? to agglutinate previously washed red cells. At the same time, all testi for rheumatoid factor activity became negative, and the anti-B isohaemagglutinin could no longer be demonstrated. The reduction of anti-B activity by absorption with solid y-globulin might indicatt. that the peculiar haemagglutinating substance and anti-B were at least partly present in the same molecules. Conforming with this v im was the observation that the agglutinating titer of serum K.G. versus washed red cells decreased significantly by absorption with group B red cells. Control absorptions with albumin treated in the same wa? as y-globulin did not reduce the different activities of serum U.G.

The different rheumatoid factor activities can be inhibited b j human y-globulin. Reaction systems with red cells coated with anti-D suitable for the demonstration of the different Gm types [lo] are particularly sensitive in this respect. In such systems, the agglutina- tion may be completely reversed by addition of inhibiting y-globulin [12, 241. In agglutination experiments with rheumatoid sera and red cells coated with incomplete anti-D, prozone phenomena are often observed, Most of these prozones can be explained by the simultaneous occurrence of a haemagglutinating substance and its specific inhibitor in rheumatoid sera [13, 311. To study whether the characteristic prozone phenomenon of serum K.G. might be explained by similar mechanisms, the following experiments were made.

Pooled human y-globulin had a marked inhibiting effect on the agglutination of washed red cells by serum K.G. Whole human serum from blood donors also had a marked effect, whereas the agamma- globulinaemic serum showed only slight inhibiting effect when used undiluted. No significant difference was observed in the inhibiting capacity of different normal sera being positive or negative for the following Gm types: Gm(a), Gm(b) and Gm(x). It has been shown above (Fig. 2) that after chromatography, fraction 4 of serum 1C.G. agglutinated washed red cells without any prozone phenomenon. Purified 7 S y-globulin from serum K.G. itself had a marked inhibiting effect on the agglutination of washed red cells by the 4th fraction. By addition of the purified 7 S y-globulin to fraction 4 and subsequent testing of the haemagglutinating capacity of serial twofold dilutions of the mixture, a definite prozone phenomenon could be produced in vitro, analogous to the in-vitro production of prozone phenomenon by mixing of Gm(a+) y-globulin with anti-Gm(a) [13]. Human

420 Harboe , R e i n s k o u , H e i s t o , B j o r n s t a d

albumin had no inhibiting effect in the concentrations tested (up to 8 g./100 ml.).

Tests for reversibility of agglutination were made as follows: In each of two tubes, group 0 red cells previously washed four times in saline were made up to a 1 per cent suspension in four ml. of serum K.G. diluted 1 : 100 in saline. After ten minutes incubation, the red cells were spun down and the supernatant carefully removed. Massive agglutination of the red cells was observed. Four ml. of saline were added t o one of the tubes, four ml. 0.6 per cent human y-globulin in saline to the other. The tubes were stoppered and carefully tilted. During one minutes tilting, the agglutination disappeared completely in the tube where y-globulin had been added, whereas it remained unchanged in the other. The cells were spun down a second time, and the absence of agglutination after addition of y-globulin was confirmed by microscopic examination.

Mechanism of Agglutination of Red Cells by Washing in Saline

The reaction between the active substance in serum K.G. and washed red cells was shown to occur very fast. Eight ml. of serum K.G. diluted 1:lOO in saline were placed in a tube in the centrifuge. One drop of a 30 per cent suspension of red cells previously washed four times was added, and the cells spun down as fast as possible. Strong agglutination of the cells was observed. This experiment made us think that the agglutination after washing of red cells previously exposed to serum K.G. might actually occur during the washing procedure because the dilution with saline then brought the system outside the prozone phenomenon. If, however, the dilution were madc with quantities of saline large enough to exceed the titer of serum K.G., no agglutination should be expected.

Table V shows the results of the experiments which confirmed this view. If the primary incubation was made with serum K.G. diluted 1:4 and washed red cells, no agglutination could take place during this phase, whereas the amount of saline added before the second phase was critical. It was established that the dilution of serum K.G. during this second phase determined the appearance or absence of agglutination. If the first incubation was made with serum K.G. diluted 1 :40, agglutination was possible during this stage. Saline was added in amounts which made the final dilution far greater than the

Studies on a Serum with Peculiar Haemagglutinating Properties 42 1

TABLE V

Mechanism of Agglutination by Washing in Saline

Serum K.G. was investigated with red cells previously washed four times in saline. Primary incubation: Two per cent red cells in recorded dilution of serum K.G. for ten minutes at room temperature. Second phase: Saline was added to the recorded final dilution of serum K.G., the cells immediately spun down, and the red cell sediment microscopically examined for agglutination. For further explanation

see text.

Dilution of Saline added Final dilution of Degree of serum K.G. during serum K.G. before agglutination primary incubation immediate centrifugation

1:4 No 1 :4 -

1:4 Yes 1:40 + -1- 1:4 Yes 1 : 400 +++ 1 :4 Yes 1 : 4000 ~

1 :40 No 1:40 -i- -t 1 :40 Yes 1 : 400 +++ 1:40 Yes 1 : 4000 I-+ t

titer of serum K.G. and the cells were immediately spun down. In this instance strong agglutination was observed.

The absorption experiments with serum K.G. and washed red cells (Table 11) and the experiments just described strongly indicated that the serum’s own 7s y-globulin inhibited the active substance from combining with the red cells. The possibility that the active substance combined with the red cells but was hindered from giving visible agglutination by surrounding y-globulin was thought less probable. From the experience of Table V, we were finally able to do an indirect Coombs’ test on cells exposed to serum K.G. One tenth ml. of serum K.G. diluted 1 :2 was incubated for 15 minutes a t room temperature with 0.10 ml. 2 per cent suspension of 0, rr red cells previously washed four times in saline. The tubes were rapidly filled with 250 ml. of saline giving a final dilution of serum K.G. of 1:5,000. The cells were spun down. After removal of the supernatant, a few ml. of saline were added, the cells transferred to a more convenient 10 ml. tube and washed two times in saline. As shown in Table VI, Coombs’ test with these cells was negative, indicating that the active substance had not combined with the red cells during the first stage of incubation.

422 H a r b o e , R e i n s k o u , H e i s t o , B j o r n s t o d

TABLE VI

lndirect Coombs' Test

Group 0, Rh -(rr) red cells were incubated with serum K.G. for 15 minutes a t 20" C. The cells were washed in saline in a manner specially devieed to avoid ag- glutination, see text. Proper controls to test reactivity of reagents are included

in the table.

Reagents Test material Coombs' Anti-7S + anti-19s Saline

serum -;-globulin R 465

0, rr cells exposed to serum K.G. Red cells coated with incomplete anti-D (ctr) i i r Red cells coated with incomplete cold

Uncoated cells (ctr) -

~

antibody (anti-H) (ctr) I + I- I t - t i -

Immunoelectrophoretic control of R 465 confirmed reactivity with 7 S and 19 S y-globulin of serum K.G.

Family Studies

Positive reactions for rheumatoid factor activity are known to occur quite frequently in asymptomatic relatives of patients with seropositive rheumatoid arthritis [25, 391. A sister, a brother, and a daughter of K.G. are living in Norway. The daughter has suffered from intermittent joint pains for some years, but there were no definite clinical signs of rheumatoid arthritis. Tests for rheumatoid factor activity were negative with sera from the three available relatives, nor did red cells exposed to these sera agglutinate by subsequent washing in saline.

Discussion

Thc observed phenomenon might be referred t o as panagglutina- tion, a term usually denoting that a serum is capable of agglutinating all red cells of the same species. This term has to be separated clearly from polyagglutinability, which refers t o the ability of the red cells of an individual t o be agglutinated by all human sera irrespective of the ABO groups involved.

The phenomenon which has been observed with serum K.G. should

Studies on a Serum with Peculiar Haemagglutinating Properties 423

be easily observed during attempts to do an indirect Coombs’ test, which is widely performed prior to blood transfusion. We have not been able to find a similar phenomenon described in the literature, and it is probably extremely rare.

Antibodies which react with “public” antigens may resemble panagglutinins. Their serological behaviour is very different from the active substance in serum K.G., and they (usually) do not react with the individual’s own cells. In autoimmune haemolytic anaemia of the warm type, the serum may contain antibodies active against all kinds of red cells tested. The patient’s red cells had a reduced survival time, but the serological behaviour of antibodies found in autoimmune haemolytic anaemia is very different from the phenomenon en- countered in the present case. Cold haemagglutinins were considered to be panagglutinins until 1956 when Wiener, Unger, Cohen and Feld- man [37] showed that they react with most human red cells but not with others, the specificity being called anti-I. Anti-I in cold haemo- lytic anaemia reacts with the patient’s own red cells [36.] All anti-I antibodies so far described have been cold antibodies; serum K.G., however, reacts equally well a t 4“ and 37” C. Anti-I specificity of the present haemagglutinating substance is further excluded by the fact that serum K.G. reacts equally well with cord blood cells as with adult red cells, whereas anti-I reacts very weakly or not a t all with cord blood cells [32]. Freiesleben and Jensen [8] described a panagglutinin active against all red cells tested including the patient’s own cells. Agglutination was observed only with cells previously washed in saline. Their phenomenon was clearly different from ours, because agglutination was inhibited by both human albumin and y-globulin, and the agglutinin could easily be absorbed from un- diluted serum by means of freshly washed red cells. Sera can be made panagglutinating by incubation with different bacteria (Davidsohn and Toharsky [4]). Stratton, Renton and Hancock [30] reported a case of pelvic abscess of long standing with polyagglutinability of the red cells and simultaneous occurrence of a panagglutinin in serum. Our patient suffered from chronic pyelonephritis. Longstanding bacter- aemia was, however, not suspected from the clinical course or autopsy findings. Bacterial contamination in uitro could be excluded, because all serum samples obtained from him were equally active and taken with sterile precautions. Weiner, Tovey, Gillespie, Lewis and Holliday [34] and Weiner and Hallum [35] have observed five sera containing a panagglutinin active in 20 per cent bovine albumin whereas no

424 H ar bo e , R eiri s k ou, H e i s t 6 , B j o r 11 s t a d

agglutination could be observed in saline. Their phenomenon is obviously different from ours.

One case quite similar to ours has been described by Heni and Blessing [18]. They found a panagglutinin in the serum of a patient with chronic lymphatic leukemia with coexistent haemolytic anaemia. When tested at 37" C., the agglutinating titer was 1: 1,024, and a prozone phenomenon was observed with no agglutination in dilution 1 : 8. Washing of red cells previously incubated with the serum resulted in agglutination provided that saline was used as washing fluid. The responsible substance could not be absorbed from undiluted serum on to washed group 0 red cells. The agglutination was inhibited by serum. These serological findings are remarkably similar to those of our case, but important differences do exist: In Heni and Blessing's case, the active substance was considerably more active a t 4" C. than a t 37" C., and the inhibition by serum was due to albumin. The agglu- tinin in serum K.G. was equally active a t 4" and 37" C., and the inhibition by serum depended on y-globulin.

A remarkable phenomenon was that red cells exposed to serum ILG. could be washed in citrate without the occurrence of agglutina- tion. The inhibition of agglutination by citrate could not be ascribed t o binding of Ca++ and/or Mg++ ions because EDTA did not prevent agglutination. Nor could the effect be ascribed to inhibition of coagu- lation factors because heparin could not prevent the agglutination. We cannot explain the effect of citrate, and more extensive investiga- tions with the intention to find other substances able to prevent agglu- tination could not be made because of scanty supply of serum. It should be mentioned that a case of cold haemagglutination has been described by Parish and Mucfarlune [26] where agglutination took place only in the presence of citrate (or oxalate) and not in saline.

We feel that the experiments showed that the active substance in serum K.G. belong to the P,-M class of proteins. The findings by absorption experiments with solid y-globulin and the different tests for rheumatoid factor activity form the basis of the conclusion that the active substance possesses some but not all of the usual rheumatoid factor activities. The lacking agglutination of washed red cells after absorption with solid y-globulin cannot be ascribed t o the simultane- ous absorption of complement [2] because heating at 56" C. (which destroys the heat labile components of complement) and the presence of EDTA (which binds Ca++ and Mg++ necessary for complement functions) did not interfere with the agglutinating ability. The in-

Studies on a Serum with Peculiar Haemagglutinating Properties 425

hibition experiments using human y-globulin also revealed reactions similar to those encountered using red cells coated with selected in- complete anti-D and anti-Gm(a) [13]. What is generally referred to as (‘the rheumatoid factor” is a t present loosely defined and usually only characterized in terms of activity by different serological tests. The responsible substances are known to be high molecular weight y-globulins with a sedimentation coefficient of 19 S, often existing in complex formation with 7 s y-globulin in the circulation [7]. The 1 9 s y-globulins arc responsible for the line usually called P,-M in immunoclectrophoresis. The factor probably consists of a multitude of different but closely related macroglobulins [15,16,17,22] occurring together in different amounts in different rheumatoid sera. The remarkable difference in reaction pattern of individual rheumatoid sera [15, 17, 331 is most easily explained in this way. Rheumatoid factor activity may be found in diseases different from rheumatoid arthritis, particularly in liver disease and some other hypergamma- globulinacmic states. I n these instances, the Waaler-Rose test is usually negative, whereas tests with human y-globulin as reactant may be strongly positive [21]. This pattern is similar t o that of our patient who had no definite signs of rheumatoid arthritis. The pathological macroglobulin of typical cases of Waldcnstrom’s macro- globulinacmia may also have rheumatoid factor activity [19]. Kunkel [20] has observed three Waldcnstrom macroglobulins with different patterns of rheumatoid factor activity. Isolated cases might thcrc- fore be expected to occur where the rheumatoid factor might show atypical reactions, possibly representing the products of antibody- producing cells slightly altered through somatic mutation, but with the pathological protein still possessing some of the specificity of the antibody-producing clone from which it was derived [23].

Most unusual is the ability of the rheumatoid factor-like protein of our patient to react with untreated red cells. By treatment of prcvi- ously washed red cells from rheumatoid arthritis patients with Dcxtran, however, Finkelstein, Kwok and Buyles [6] claimed t o detect the rheumatoid factor as a coating of the patient’s red cells in a large percentage of cases. Rheumatoid arthritis serology and blood group serology were brought together by the detection of inheritable serological differences among human y-globulins [9, 101. The present case presented unusual problems during compatibility tests prior to blood transfusion, which could only be solved by serological techniques usually involved in rheumatoid arthritis sero-

426 H a r b o e , Reinskou, Heis to , B j o r n s t a d

logy. If it turns out t o be true that the rheumatoid factor often combines directly with untreated red cells, the connection might be even closer than hitherto assumed.

Acknowledgements

The authors are indebted to Dr. K. Arnesen of Department of Pathology, UllevHl Hospital, for provision of data from post-mortem examination and to Dr. J. Lundevall for valuable advise concerning preparation of the manuscript.

Summary

The serum of a patient suffering from general arteriosclerosis and chronic pyelonephritis with mild anaemia was investigated because of difficulties during compatibility tests prior to blood transfusion. Red cells exposed to the serum were strongly agglutinated when subsequently washed in saline. The responsible sub- stance was identified as a y-globulin of high molecular weight, also possessing some but not all of the usual rheumatoid factor activities.

Zusammenfassung

Wegen Srhwierigkeiten bei der Kreuzprobe wurde das Serum eines Patienten mit generalisierter Arteriosklerose, chronischer Pyelonephritis und leichter Anamie niiher untersucht. Erythrozyten, die in diesem Serum aufgeschwemmt waren, wur- den beim nachfolgenden Waschen in physiologischer Kochsalzlosung kraftig ag- glutiniert. Die fur dieses Phanomen verantwortliche Substanz erwies sich als hoch- molekulares y-Globulin, welches einige, jedoch nicht alle Eigenschaften des Rheuma- faktors aufwies.

R6sunt6

Le serum d'un patient atteint d'artCriosclCrose g6nBralisBe et de pyelonkphrite chronique avec andmie a 6th examine par suite des difficult& que ce &rum prBsen- tait lors des Bpreuves de compatibilitb pratiquees en vue de transfusions. Les 6ry- throcytes mis en contact avec ce sBrum Btaient fortement agglutinhs lors du lavage en milieu salin. La substance responsable a B t B identifiee comme Btant une gamma- globuline de haut poids mol6culaire qui possedait un certain nombre, mais pas toutes les propriBtBs habituelles du facteur rhumatoide.

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Author’s address: Dr. M. Harboe, Institute for Thrombosis Research, Rikshospitalet, Odo (Norway)