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The role of antinuclear reactions in the diagnosis of systemic lupus erythematosusA study of 53 cases.

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Arthritis and Rheumatism
JUNE, 1961
VOL. IV, NO. 3
The Role of Antinuclear Reactions in the Diagnosis of
Systemic Lupus Erythematosus:A Study of 53 Cases
Fifty-three patients with symptoms suggestive of S.L.E.were studied clinically
and serologically (antinuclear reactions
included complement fixation against
nucleoprotein, DNA, and histone, tests
for rheumatoid arthritis). Clinically and
serologically, the patients with (72 per
cent) and without (18 per cent) L.E.
cell phenomenon did not differ significantly and seem to represent one nosological entity. It is suggested that in
the past too much emphasis has been
placed on a positive L.E. cell test in
establishing the diagnosis of S.L.E. The
results clearly indicate that in addition
to this test, antinuclear reactions are an
indispensable tool in establishing the
diagnosis of S.L.E.
Un serie de 53 patientes con symptomas
suggerente systemic lupus erythematose
esseva studiate clinica- e serologicamente. Le reactiones antinucleari includeva fixation de complemento contra
nucleoproteina, acido disoxyribonucleic,
e histona e tests de arthritis rheumatoide.
Le patientes con e illes sin le phenomeno
de cellulas L.E. (72 e 18 per cent) non
differeva significativementeab le punctos
de vista clinic e serologic. I1 pare que
illes representa le mesme entitate nosologic. Es presentate le opinion que in
le passato troppo importantia esseva attribuite a1 positive test de cellulas L.E.
in le establimento del diagnose de syskmic lupus erythematose. Le resultatos
indica clarmente que, a parte iste test,
reactiones antinucleari es indispensabile
in establir le diagnose de systemic lupus
erythematose.
C
HTERIA FOR THE DIAGNOSIS of systemic lupus erythematosus (S.
L.E.) have been constantly modified since its recognition by Kbposil and
Osler.2 Until the discovery of the L.E. cell phenomenon the disease was believed to be extremely rare, acute and rapidly fatal. With this test, a highly
specific diagnostic criterion was added to the more or less well defined clinical
description of S.L.E. It soon became apparent that the clinical concept of the
disease needed revision, since many patients showed symptoms which had not
previously been considered to be characteristic of the disease. On the basis of
the L.E. cell phenomenon, the clinical entity S.L.E. seemed well defined and
From the Department of Medicine and the Rheumatic Diseascs Study Group, New
York Unioersity School of Medicine, and the Third Medical Division, Bellewe Hospital
Catter.
Aided b y U.S.P.1f.S.grants #A-1431 (C-3), A-2360 (C-11,
and 4-3777,and by a grant
from The Nutiom1 Foundation.
223
224
ROTHFIELD, PHYTHYON, M C EWEN AND MIESCHER
most of the “false positive” L.E. cell tests were soon recognized as morphologically different from the “genuine” L.E. phenomenon. Thus, the pseudo-L.E.
phenomenon in many hypersensitivity and other disorders is characterized by
the phagocytosis of nuclear material which still shows some chromatin pattern and which is usually as basophilic as the nucleus of the phagocytiu’ng
ce11.3 One exception which has been accepted by all authors is the L.E. cell
phenomenon in hydralazine toxicity, in which typical L.E. cells are usually
found. After withdrawal of the drug, the L.E. cell test becomes negative and
the symptoms usually disappear.
Thus, in the first years after the introduction of the L.E. cell test, S.L.E.
seemed a relatively well defined entity which could easily be distinguished
from related collagen diseases on the basis of this biological reaction, and patients with rheumatoid arthritis and positive L.E. cell tests were simply diagnosed as S.L.E. With more careful clinical analysis, however, it was recognized
that the problem was more complicated, i.e. that a positive L.E. cell test in a
patient with rheumatoid arthritis does not necessarily mean that the patient
has S.L.E.4-6Moreover, in rare patients with liver disease, a positive L.E. cell
test was also found.’ With the finding that the L. E. cell phenomenon is not as
specific for S.L.E. as was previously thought, diagnostic difficulties again
arose.
Since 1953 a number of serological reactions have been developed to detect
the L.E. factor and other antinuclear factors.*-ll The same series of events occurred: after an initial optimistic period in which it was hoped that a more
specific method for the diagnosis of S.L.E. had been developed, it was recognized that no one of the antinuclear reactions was absolutely specific for the
disease. However, on the basis of these new immune biological reactions, it
has become apparent that a variety of different clinical syndromes belong to
the same family of disease.
With these new diagnostic tools the question recurs as to how S.L.E. should
be clinically diagnosed. This paper deals with this problem on the basis of
a clinical and serological study of 53 patients. Thirty-eight patients had clinically typical S.L.E. with positive L.E. cell tests. A second group of 15 patients
were suffering from a chronic disease suggestive of S.L.E. but with repeatedly
negative L.E. cell tests. Not included in this group are patients with clinically
well-defined collagen diseases such as dermatomyositis, systemic scleroderma,
periarteritis nodosa and rheumatic fever. Patients with uncomplicated rheumatoid arthritis are not included in this study.
The patients have all been observed for a period varying from six months to
ten years. The serological studies were carried out during 1959 and the first
months of 1960.
MATERIALS
AND METHODS
Patients.--Group I-Thirty-eight patients in whom the diagnosis of S.L.E. was made
on the basis of typical multiple system involvement. In all patients L.E. cells were found
in preparations of peripheral blood.
Group 11-Fifteen patients in whom the diagnosis of S.L.E. was made on the basis of
typical multiple system involvement. L.E. cells were not found either in multiple prepata-
ANTINUCLEAR REACTIONS IN SYSTEMIC LUPUS ERYTHEMATOSUS
225
tions of peripheral blood or in bone marrow.
All patients were observed personally by one of us.
Serum-Most sera from patients were tested within a few days after collectioii. Others
were stored at --u) C. for periods ranging from me week to several months.
L.E. cell test.-Slides were prepared by the method of Zimmer and Hargraves.12
Conglutinin complement fixation test.-Complement fixation was measured by the use
of the highly sensitive conglutination system, consisting of bovine serum (as a source of
natural anti-sheep erythrocyte antibody and conglutinin) and sheep erythrocytes. In the
presence of complement, agglutination of the sheep erythrocytes occurs. If a complement
fixing reaction takes place between a patient’s serum and an appropriate antigen, the conglutination reaction is inhibited,
1. Bufier.-All the dilutions were made using the following buffer ( p H = 7.4): three
parts 0.2 M Na,HPO,, one part 0.2 M NaHpPO,, six parts H,O, and thirty parts 0.15 M
NaCl.
2. Sheep erythrocytm-Sheep erythrocytes (less than 10 days old) were washed three
times in phosphate buffer and then centrifuged at 2400 rpm for five minutes. Eight-tenths
ml. of the packed erythrocytes were then suspended in 19.2 ml. of buffer ( 4 per cent suspension )
3. Bovine serum.-Bovine serum was first inactivated at 56 C. for 30 minutes. The dilution to be used in the complement fixation test was determined by titrating the bovine
serum (twofold dilutions), against various dilutions of complement (fresh human serum,
1.25-fold dilutions), using inactivated human serum as a control. The following were placed
into depressions of a translucent plastic tray: 0.05 ml. of diluted bovine serum, 0.05 ml. of
sheep erythrocyte suspension, 0.05 ml. of diluted Complement. 0.10 ml. of buffer. The
plastic tray was covered with moist cardboard and incubated at room temperature for 30
minutes. Agglutination of erythrocytes was graded from one to four plus. Bovine serum
which caused agglutination of erythrocytes in the presence of any dilution of inactivated
human serum was discarded. The optimum titer of bovine serum was that which gave
three to four plus agglutination with the highest dilution of complement. Good sen usually
showed titers of 1:80 or more. Bovine sera can be stored at 4 C. for periods of weeks.
Frozen at --2o C., it keeps its activity for years.
4. Conglutinin system.-The conglutinin system was prepared by mixing the 4 per cent
sheep cell suspension with an equal amount of bovine serum in the dilution found adequate
in the previous experiment. The final concentration of sheep erythrocytes in the conglutinin system is thus 2 per cent.
5. Comp~ement.-O.O5ml. of 1.25-fold dilutions of fresh human serum, 0.10 ml. buffer,
and 0.10 ml. of conglutinin system were placed in depressions in the plastic tray and left
at room temperature for 30 minutes. The tray was slightly shaken every five minutes. Complement was used at twice the concentration which produced clear-cut agglutination of
the sheep erythrocytes. This titer was at least 1:16, in which case complement would be
used at a titer of 1:8.
6. Antigens.-Calf
thymus nucleoprotein was supplied by Hormon Chemie, Munich
(prepared according to Dounce, J. Biol. Chem. Am., 147:151, 221, 235, 683, 1943; Mirsky
and Pollister, Biol. Symp. 10:247,’1943). A fine suspension of 100 gamma/ml. was prepared prior to use. This suspension usually became anticomplementary if kept for a prolonged period of time at 4 C. Calf thymus DNA was supplied by Mann Research Laboratories, Inc., New York, N.Y. It was used in a concentration of 20 gamma/ml. Calf thymus
histone was supplied by W o r t h i n e n Biochemical Corporation, Freehold, N. J. This
preparation contained a small amount of insoluble material which was strongly anticomplementary. A 100 gamma/ml. suspension of histone was prepared immediately prior to
each test. The insoluble particles were eliminated by centrifugation at 3,000 rpm for 30
minutes. After this procedure, the histone solution was no longer anticomplementary, but
would again become anticomplementary if stored for a prolonged period of time.
All antigen solutions were tested for anticomplementary activity prior to use.
.
226
ROTHFIELD, PHYTHYON, MC EWEN AND MIESCJ3m
7. Technic.-The following solutions were placed in depressions in a plastic tray: 0.05 ml.
of twofold dilutions of the patient’s serum, 0.05 ml. antigen solution, 0.05 ml. of complenient.
Controls were prepared using antigen without patient’s serum and patient’s serum without
antigen, and complement without antigen and antiserum. The tray was covered with moist
cardboard and incubated overnight at 4 C. Complement dilutions were simultaneously incubated on a separate tray. If, on the following morning, the non-specific Complement titer
loss was more than 50 per cent, an appropriate quantity of complement was added to the
test system. One-tenth ml. of the conglutinin system was then added. The tray was slightly
agitated every five minutes and carefully observed for the appearance of agglutination. If
complement had been fixed, no agglutination occurred, whereas the lack of complement
fixation was demonstrated by the agglutination of erythrocytes. The results were compared
with the antigen and serum controls. Anticomplementary serum showed no agglutination
in the serum control. Serum containing heterophile antibodies gave very strong reactions
with higher serum concentrations. This interfered with the fixation of complement, and
such sera were therefore first absorbed with sheep erythrocytes.
Tests for rheumatoid factor.-The latex fixation test was done according to the method of
Singer and Plotz.13 Sheep erythrocyte agglutination (SEA) and inhibition tests were performed by the method of Ziff et al.14
rabbit antihuman gamma globulin
Fluorescent antibody test.-Fluorescein-conjugated
(Cohn Fraction 11) was prepared according to the method of Coons and Kaplan.15 Fluorescein isothiocyanate’ was added as the dry powder to the reaction mixture. The fluoresceinconjugated antiserum was absorbed twice with acetone extracted guinea pig liver powder
immediately before use (0.2 grams liver powder for the first and 0.1 grams for the second
absorption of 2 ml. of conjugate). The counterstaining procedure, as described by Smith et
al.,l6 was performed by adding 20 per cent Lissamine-Rhodamine RR 2,001 conjugated whole
rabbit serum to the antiglobulin serum. Lyophilized nuclei isolated from guinea pig splccn,’7
were used as the antigen. Three mg. of lyophilized nuclei were suspended in 1.0 ml. of
distilled water. Small drops of these suspensions were allowed to dry on microscope slides.
They were then fixed in 95 per cent ethanol at room temperature for 30 minutes and stored
in the cold ( 4 C. ) . In general, results were better if these fixed nuclei were used within 24
hours. There appeared to be little difference in staining affinity between various types of
nuclei (calf thymus, mouse liver, etc. ). However, when the fluorescent antisera were absorbed with acetone extracted guinea pig liver, guinea pig nuclzi exhibited less non-specific
staining in the control slides. Two small spots of nuclei were placed on one slide, so that
each slide could be used as both test and control with normal human serum. The nuclear
spots were covered with undiluted serum. The slides were then incubated in a moist
chamber for 20 minutes. The slides were washed for 10 minutes in each of two changes
of 0.01 M phosphate buffered saline ( p H 7.1). The nuclear spots were covered with the
fluorescent antihuman rabbit globulin for 20 minutes, washed again twice as above, and
mounted in diluted buffered glycerol. The preparations were examined in U.V. light and
graded according to degree of nuclear fluorescence. A known negative and positive serum
were included in each group tested. A Zeiss “Ultraphot” microscope was used with an
Osram HBO 200 lamp and a dark field condenser. The light was filtered with a BG 12
exciter filter and an OG 5 barrier filter. The nuclei were examined for fluorescence under
oil immersion. Under these conditions, the unstained (control) nuclei exhibit a dull yellowish orange fluorescence. Those of the positive sera showed the specific bright green
color of fluorescein.
RESULTS
Clinical Data
The two groups of patients (Group I: clinical diagnosis of S.L.E. and
positive L.E. cell test; Group 11: clinical data suggestive of S.L.E. but repeated‘Supplied by Nutritional Biochemical &., Cleveland, Ohio.
+Supplied by Arnold, Hoffman and Co., Providence, R. I.
227
ANTINUCLEAR REACTIONS IN SYSTEMIC LUPUS ERYTHEMATOSUS
ly negative L.E. cell tests) showed no difference with respect to sex, age of
onset of the disease, and race (table 1). In both groups, females were more
frequently involved than males. The age of onset of the first symptom was
between 8 and 80 years in Group I and 18 and 45 years in Group 11, with the
similar averages of 28 and 33 years respectively. All 53 patients had been treated
with adrenal steroids at some time during the course of their illness and all
responded well to this therapy.
There was no significant dihrence between the two groups in duration of
disease from the onset of the first symptom (table 2). It should be stressed that
the average time of observation was significantly shorter in Group 11. However,
some patients in Group I1 have been observed for as long as 10 years with
repeatedly negative L.E. cell tests, as illustrated by thg following case report.
L.S., a white female, became ill for the first time at the age of 23 (1949).Ten days
after taking a penicillin lozenge for a sore throat she developed fever, urticaria, arthritis,
severe anemia and thrombocytopenia. One year later, in March 1950, a sore throat (not
treated with penicillin) was followed by urticaria, an erythematous rash on the arms and
legs, and arthritis involving knees, ankles, and small joints of the hands. She was admitted
to the Third Medical Division of Bellevue Hospital, where she was found to have, in addition, lymphadenopathy. Bone marrow and muscle biopsy were normal. No diagnosis was
made. During the next year she had three similar acute, febrile episodes requiring hospitalization. L.E. cell tests were always negative. During her fifth admission in 1955, positive
sheep erythrocyte agglutination and inhibition tests were found. At this time steroid
therapy was started. In August 1956, after withdrawal of steroids, erythematous rash on
the extremities developed after sun exposure a t a beach. This was followed by fever, chills,
conjunctivitis, and muscle tenderness. Muscle biopsy was again normal. Since that time
she has been maintained on prednisone. There has been persistent mild arthritis, with deformities of the small joints of the hands, knees and wrists. The erythrocyte sedimentation
rate (Westergren) varies between 25 and 112 mm. in one hour. Sun exposure has been
conscientiously avoided. Twenty-five L.E. cell tests have been done since 1950 and all
have been negative. In 1959 fluorescent antibody test was negative; antibodies to DNA
and nucleoprotein were demonstrated.
Table l.--Sex, Race
and
Age at Onset
Group J
Group I1
38
34
4
15
13
2
28
33
15
14
8
1
10
4
1
0
Total number of patients
Females
Males
Age at onset of first symptom
(average in years)
Race
White
Negro
Puerto Rican
Chinese
Table 2.-Duration of Disease
and
Length of Observation
Duration of Disease.
Menn
Standard Deviation
Group I
Group I1
.Years.
6.4
6.0
3.78
4.93
Length of Observation.
Mean
Standard Deviation
3.34
2.10
2.32
1.89
228
ROTHFIELD,
PHYTHYON, MC EWEN
AND MIESCHER
Comment: This patient showed a characteristic clinical course of S.L.E.
There was involvement of skin, joints, eyes, and blood (anemia and thrombocytopenia). The rash was not diagnostic of lupus. On the basis of these data,
the diagnosis of S.L.E. is suggestive despite negative L.E. cell tests. However,
the clinical course together with the demonstration of antibodies against
nucleoprotein and DNA, establishes the diagnosis on a firm basis.
Organ System Involvement
1. Joints.-Arthritis was the most frequent manifestation of the disease in
both groups. Joint involvement was most commonly polyarticular and usually
localized in the small joints of the hands. In a few patients arthritis was present
in only one or two of the larger joints. Deformities occurred in approximately
one-third of all patients in each group. However, the incidence of deformities
in patients with arthritis was somewhat higher in Group 11. Primary arthralgia
without evidence of arthritis was uncommon, although acute polyarthritis was
frequently followed by arthralgia during periods of remission.
2. Skin.-In addition to the “typical” erythematous rash, skin involvement
included sun sensitivity, alopecia, and urticaria. The two groups were similar
in frequency and distribution of the rash. An isolated ‘‘butterfly’’ lesion was an
uncommon manifestation of the disease in both groups. Residual areas of hyperpigmentation were present in approximately one-half of the patients in
each group and occurred in Negro, White and Chinese patients. Sun sensitivity
was also present in approximately one-third of the patients in both groups.
Alopecia was more common in Group I. Hair loss was both diffuse and patchy
in distribution and occurred during exacerbation of the disease in both groups.
Permanent areas of alopecia were present in a few patients in each group and
were located in scarred areas of the scalp which had previously been involved
by an erythematous rash.
3. Kidney.-Renal involvement was more common in Group I than in Group
11. Proteinuria was the predominant finding in both groups. The percentage
of patients with nephrotic syndrome was similar in both groups. All patients
with renal disease were found to have urinary abnormalities already present
at the time of first observation. Renal tissue was obtained from post-mortem
material or by percutaneous renal biopsy in nineteen patients in Group I and
in two patients in Group 11. Results of pathological study of the patients in
Group I, in addition to others, will be reported in detail elsewhere.]*
In Group I, two patients with no clinical evidence of renal disease had normal
kidney tissue. Tissue from three other patients without clinical renal disease,
and from seven patients with mild renal disease ( hematuria and albuminuria),
showed only basement membrane thickening and focal hypercellularity of
glomeruli. Tissue obtained from patients during exacerbation of the generalized
disease showed, in addition to thickening of the basement membrane and
focal hypercellularity, focal necrosis of glomeruli. Seven patients with renal
insufficiency had the changes described above in a more diffuse pattern. In
addition, most had crescents, wire-loops, or hyalinization of glomeruli. Hematoxylin bodies were seen in glomeruli in only one patient who died with
nephrotic syndrome and renal insufficiency.
ANTINUCLEAR REACTIONS I N SYSTEMIC LUPUS ERYTHEMATOSUS
229
Both patients in Group I1 in whom kidney tissue was obtained had basement membrane thickening. One patient had mild renal disease. Focal and
diffuse glomerular hyalinization was present at post mortem examination. The
other patient died from renal insufficiency. Pathological examination revealed
qlomerular infiltration with neutrophilic leukocytes and occasional focal necrosis in glomeruli. Medium sized arteries were found to have organized or
organizing thrombi with partial destruction of their walIs. These findings were
suggestive of periarteritis nodosa. Hematoxylin bodies and wire loops we=
not seen in the kidney tissue obtained from these two patients in Group 11.
4. Lung and P2eura.-More than one-third of the patients in Group I had involvement of the lung or pleura in contrast to one-fifth of Group 11. Only patients in whom fluid and/or infiltrate was demonstrated by X-ray are included,
although many more patients in both groups complained at times of pleuritic
type chest pain. Infiltrates in the lung parenchyma occurred approximately as
often as pleural effusions. The infiltrate characteristically did not clear with
antibiotic therapy, but disappeared when steroids were given in sufficiently
high dosage. One patient in Group I1 had pulmonary fibrosis and is reported
in detail e1se~here.l~
5. Cardiac.-Pericarditis occurred in approximately 10 per cent of patients
in both groups. Electrocardiographic abnormalities occurred in nearly onethird of the patients in each group, and consisted most frequently of nonspecific T-wave changes. Other electrocardiographic abnormalities were prolongation of the Q-T interval, incomplete right bundle branch block, prolongation of the P-R interval, and elevation of the S-T segment. Significant
(louder than grade I ) apical svstolic murmurs were heard in only four patients in Group I. In addition, two of these patients had transient apical diastolic murmurs during the course of their illness.
6. Blood Vessels.-Vascular involvement, including absent pulses, thrombosis, and Raynaud‘s syndrome, occurred in approximately one-fourth of the patients in both groups. Absent pulszs were found on physical examination in onefifth of the patients in both groups, including many who had no symptoms referable to the vascular system. Raynaud’s syndrome was present in a similar
percentage of patients in each group. A patient in Group I1 who had severe involvement of the vascular system is presented below.
M. G., a white female, had been well until January 1950, at age 48, when she was
treated with penicillin for a sore throat. Ten days later the patient developed acute polyarthritis and fever. In April 1950 she developed a pleural effusion, a vacation was advised and the patient went to Florida, While in Florida, fever, exacerbation of the arthritis,
and severe chest pain occurred and, on returning to New York, the patient sought admidon
to Bellevue Hospital. Physical examination revealed polyarthritis, left pleural &sian apd
pericarditis. Results of routine laboratory tests were normal and many L.E. cell tesb were
negative. She was treated with 50 mg. of prednisone and responded well. In July 1956 a
pruritic erythematous rash appeared in the ‘%utterfly” area of the face. In September 1956
the patient noted pain and blanching of the fingertips in cold weather and was admimed
for the second time in October 1950 because of multiple infected and gangrenous areas on
the fingertips. The other fingers on both hands were found to be cold. white, and tend-er
to touch. Results of routine laboratory tests were negative and four L.E. cell tests wexe
negative. She was treated with aspirin, dibenzyline and a stellate ganglion block with relief
of pain and healing of the lesions.
230
ROTHFIELD, PHYTHYON, MC EWEN AND MIESCNER
Since 1958 the patient has had intermittent arthritis involving knees, wrists, shoulders and
small joints of the hands, and Raynaud's syndrome during the winter. She has been maintained on small doses of prednisone (2.5 to 5 mg. ). Eleven L.E. cell tests since discharge
from the hospital have been negative. Fluorescent antibody test was positive. Complement
fixation test was positive for nucleoprotein and negative for histone and DNA.
7. Eyes.-Conjunctivitis was the most common eye manifestation in Group I,
occurring in eleven patients. It occurred during exacerbation of other manifestations and subsided with systemic steroid therapy. Frequently an exacerbation of the disease involving other organs was preceded by conjunctivitis. Only
two patients in Group I1 had conjunctivitis. Cytoid bodies were seen in four
patients in Group I, although all patients had fundoscopic examination at the
time of acute illness. Cytoid bodies were transient, disappearing during remission. Retinal hemorrhages and exudates in a few patients with severe renal
insufficiency and hypertension in each group have not been included as ocular
manifestations of the primary disease.
8.Ob-Gyn.-Temporary amenorrhea during exacerbations was the most common abnormality, with normal menses often returning during remissions. Three
of nineteen pregnancies occuring during the course of the disease terminated
in a stillbirth or miscarriage in Group I. One pregnancy of five terminated in a
miscarriage in Group 11. A small number of patients in each group complained
of exacerbation of symptoms just prior to or during menses.
9. Liver, Spleen and Lymph nodes.-Lymph node enlargement was slightly
more common in Group I. On the other hand, enlargement of the spleen occurred three times more often in Group 11. Liver enlargement, present in about
one-third of all patients, never extended more than three to five centimeters
below the right costal margin. In a few patients in both groups the liver was
slightly tender. One patient in Group I1 had obstructive jaundice due to carcinoma of the head of the pancreas.
10. Peripheral neroous system-Involvement of the peripheral nervous system consisted of nerve palsies which were transient, lasting from one to six
weeks, The nerve most frequently involved was the trigeminal. A similar percentage of peripheral nervous system involvement was present in Group I and
Group 11. Unlike other organ systems, the peripheral nervous system was frequently involved at a time when the patient was otherwise in remission.
11. Central Nervous System-Involvement of the central nervous system
( excluding organic mental syndrome ) was manifested by convulsions, severe
localized headaches, hemiparesis, and hemi-chorea. Approximately one quarter
of the patients in Group I had central nervous system involvement, whereas
none in Group I1 exhibited any of the above manifestations. Convulsions (nonuremic) were most common and, with one exception, occurred in patients with
organic mental syndrome.
12. Psychosis.-Psychosis occurred in slightly more than one-third of the
patients in both groups. Organic mental syndrome was the more common type
of psychosis in both groups. One-third of the patients were seen on the medical
service of the Bellevue Psychiatric Hospital. This undoubtedly explains the
higher percentage of psychosis in these patients than in other series in which
231
ANTINUCLEAR REACTIONS IN SYSTEMIC LUPUS ERYTHEMATOSUS
an incidence of 9 to 17 per cent has been reportedS2O22 The patients reported
here are included in a larger series of patients with psychosis reported elsewhere in detai1.23
Laboratory Data
Routine laboratory data are shown in table 3. Anemia was present in more
than one-half of the patients in each group. In all cases, the anemia occurred
immediately before or during an exacerbation of other manifestations of the
disease and clinical remission was followed by a return of the hemoglobin to
low normal or normal level. One patient in each group had a hemolytic anemia
with positive Coombs test. Marked leukopenia occurred in a few patients in
each group. Thrombocytopenia of less than 100,000/mm.3 was slightly more
common in Group 11. During the course of their disease approximately one-half
of the patients in Group I had a slight diminution of the platelet count (120,OOO
-180,000/mm.3). All patients in both groups had elevated erythrocyte sedimentation rates during the course of their illness. However, a small number
of patients in each group had normal sedimentation rates during remission.
False positive serological tests for syphilis with negative treponema immobilization tests occurred in approximately one-quarter of the patients in both
groups.
The results of three tests for rheumatoid factor are shown in table 4. The
incidence of rheumatoid factor was similar in the two groups. The inhibition
test gave the highest percentage of positive results in each group. The rheumatoid factor was present more frequently in those patients with chronic deforming arthritis. On the other hand, the rheumatoid factor could not be demonstrated in one patient with severe rheumatoid-like deformities in Group I.
Antinuclear Reactions: The results of the antinuclear reactions are s h o w in
table 5. Three patients in Group I and four patients in Group I1 could not be
tested by the complement fixation method because their sera were anti-complementary. Of the three patients in Group I with anti-complementary serum,
two had positive fluorescent antibody tests. Three of the four patients in Group
I1 with anti-complementary serum had positive fluorescent antibody tests. The
patient in Group I1 with negative reactions to all antinuclear tests is described
below.
Table 3.-Hematologic Abnormalities
Abnormalities
Anemia (hemoglobin less than 10
grams per cent)
Leukopenia (less than 3,000/mms)
Thrombocytopenia (less than
100,000/mm3
Elevated erythrocyte sedimentation
rate (more than 30 mm. in one
hour-Westergren)
False positive serological test for
fiy-philis
Group I
Number of
Patient7
%
Group I1
Number of
Patients
74
22
3
58
8
8
1
53
5
13
4
27
38
100
15
100
R
31
4
7
27
232
ROTHFIELD, PHYTHYON, MC EWEN AND MlESCHER
Table 4.-Results
of Tests for Rheumatoid Factor
Latex Fixation
Pos.
N~s.
Patienta with
Group I
No joint symptoms
Transient joint symptoms
Non-deforming chronic
arthritis
Chronic deforming arthritis
Total
Group I1
No joint symptoms
Transient joint symptoms
Non-deforming chronic
arthritis
Chronic deforming arthritis
Total
0
0
S.E.A. Agglutination
POS.
Neg.
1
9
2
7
9
7
26
9
I
0
1
0
0
9
1
8
0
1
0
1
1
8
-3
6
6
J
"
6
7
S.E.A. Inhibition
Pos.
Neg.*-
2.5
7
11
21
1
0
0
1
0
1
0
1
0
3
4
3
3
I
2
0
9
I
4
:3
3
3
4
7
4
7
7
'Positive test.
Table 5-Antinuclear
~
Fluoreseent
Antibody
Pos.
Neg.
Group I
Group I1
Rheumatoid
Arthritis
Control random serum
33
10
5
Reactions
~~~~~~~
Conglutinin-ComplementFixation
Nueleoprotein
Pos.
Neg.
____
Pos.
DNA
Neg.
Histone
Pos.
Nea.
Antieomplementary
8-m
.___-______
5
23
6
12
5
18
4
17
7
S
0
11
3
4
5
2,5
2
37
2
37
1
.%
0
2
98
6
194
0
200
8
192
0
' G. S., a white female, was well until the age of 28 (1950),when she developed an
erythematous, thickened rash in the butterfly area of the face. The lesion was diagnosed
as discoid lupus erythematosus after a skin biopsy was performed. A few years later similar
lesions appeared in the scalp which finally healed, leaving small areas of permanent alopecia.
At age 32, morning stiffness of the knees and elbows began and one year later objective
arthritis of these joints with intermittent fever was present. Since that time she has been
treated intermittently with adrenal corticosteroids with good response of both the arthritis
and the rash. However, at present there is crepitation and limitation of motion of the right
knee and right elbow. X-rays of these joints show loss of joint space with marked osteoporosis of the adjacent bone. All laboratory studies, inclitding tests for rheumatoid arthritis
and all antinuclear reactions, have been negative, except for a persistently elevated erythrocyte sedimentation rate. T h e patient's father was seen by one of us in a Veterans Administration Hospital where he was being treated for severe deforming rheumatoid arthritis.
Comment: In this case with negative L.E. cell tests and with negative reactions to all antinuclear factors, only two systems were involved, i.e. joints and
skin. However, the skin lesions were typical of lupus erythematosus both clinically and histologically. The clinical course does not d i h r from that of other
paiients with only skin and joint involvement and positive L.E. cell test. Therefore the diagnosis of S.L.E. in this patient seems probable.
DISCUSSION
A group of 38 patients with the clinical diagnosis of S.L.E. and with positive
L. E. cell tests (Group I ) has been compared to a group of 15 patients with a
233
ANTINUCLEAR REACTIONS IN SYSTEMIC LUPUS ERYTHEMATOSUS
clinical syndrome consistent with S.L.E. but with repeatedly negative L.E.
cell tests (Group 11).
The duration of the disease was similar in both groups, although patients
in Group I were observed for a longer period of time. In general, patients in
Group I were more severely ill and sought medical care earlier in the course
of their disease. The extent of the disease is indicated by the number of organ
systems involved (fig. 1).Patients in Group I1 had an average of 4-6 systems
involved as contrasted to 6-8 systems in Group I. If the various organs are considered separately (table s),a more frequent involvement of joints, skin, kidney, lung, and pleura, and central nervous system was found in Group I. In
addition, involvement of heart, lymph nodes and eyes was somewhat more frequent in Group I. Only the incidence of splenomegaly was higher in Group 11.
Laboratory data (table 3) do not show any significant differences between the
two groups.
It has been assumed that the immune biological reactions reflect the pathogenesis of the disease either directly or indirectly. For this reason, the results
of these reactions might indicate whether the two groups comprise one or two
disease entities.
The disease most closely related to S.L.E. seems to be rheumatoid arthritis.
Whereas the differential diagnosis in any one patient may be difficult, the
immunological data are quite digerent in the two diseases. Antinuclear antibodies are found much more frequently in S.L.E. and the rheumatoid factor is
present more frequently in rheumatoid arthritis. If Group I included a considerable proportion of patients with rheumatoid arthritis, one would expect
an immune serological pattern typical of this disease. As seen in table 4, this
is not the case. The SEA inhibition test, which is the most sensitive test for
rheumatoid arthritis, and which is positive in over 95 per cent of patients with
rheumatoid arthritis14is positive in 33 per cent of the patients in Group I1 and
29 per cent of patients in Group I. Thus, it is obvious that Group I1 does not
have the serological pattern typical of rheumatoid arthritis. We may thus conNUMBER
OF
PATILNTS
I
0
= aaom
I
a
=
II
GROUP
5
2
NuueEa
3
OF
4
5
6
7
9
10
11
I2
sYsiEus IWVOLVIO
Fig. 1.-Number of organ systems involved in patients in Group I and Group 11.
Organ systems: joints, skin, kidney, lung, heart, blood vessels, eyes, ob-gyn., liver,
spleen, lymph nodes, central nervous system, peripheral nervous system, psychosis,
blood (anemia, leukopenia, or thrombocytopenia)
I
234
ROTHFIELD, PHYTHYON,
Table
&-Organ
Stlstem Znvolvement
Group I
Joints
Skin
Kidney
Lung and Pleura
Cardiac
Blood Vessels
Eyes
Ob-Gyn
Liver Enlargement
Splenomegaly
Lymphadenopathy
Peripheral Nervous System
Central Nervous System
Psychosis
MC EWEN AND MIESCHER
Group I1
NO.
"0
36
32
27
15
20
11
13
15
13
4
95
10
84
10
8
22
7
9
14
71
39
51
29
NO.
3
vn
66
86
53
20
40
-34
6
4
2
39
34
5
12
58
18
5
6
2
27
13
15
33
33
40
13
24
37
0
6
0
40
3
clude that Group I1 does not represent patients suffering primarily from rheumatoid arthritis.
The incidence of positive antinuclear reactions in the two groups differs only
slightly. It must be stressed that only 1 patient in each group had negative results with all antinuclear serological tests, i.e., fluorescent antibody and complement fixation with nucleoprotein, DNA and histone. -4n additional patient
in Group I had a negative fluorescent antibody test and anticomplementary
serum so that complement fixation tests could not be performed. These three
patients do not differ clinically from other patients in the two groups. The immune serological patterns of the two groups of patients seem to differ only
quantitatively and would support the conclusion that we are dealing with a
single homogeneous group with only quantitative differences.
It is our general experience that milder clinical forms of S.L.E. usually show
weaker immune serological reactions. Among the antinuclear reactions, antibodies against DNA disappear most frequently in spontaneous or drug induced
If the clinical state of the patients in Group I is considered in
rerni~sions.~~
relation to the presence of antibodies to DNA, there were 60 per cent positive
reactions during an exacerbation of the disease and only 45 per cent positive
reactions in patients who were in remission. The 36 per cent positive reactions
in Group I1 correlates with the generally milder disease in these patients.
If the immune biological reactions reflect directly or indirectly the pathogenetic mechanisms of the disease, it might be possible to correlate the presence of antinuclear antibodies with the involvement of particular organ systems. Such a correlation, indeed, does exist between the rheumatoid factor and
involvement of the joints. The rheumatoid factor was present predominantly
in patients in both groups who had chronic deforming arthritis. The antinuclear antibodies, on the other hand, could not be related to involvement of
any particular organ system. For example, complement fixation tests with
nucleoprotein were positive in 61 per cent of patients with arthritis and in 88
per cent of patients with renal disease. The same lack of organ specificity was
found when nucleoprotein, DNA and histone antibodies were correlated with
involvemmt of other organ systems,
ANTINUCLEAR REACI'IONS IN SYSTEMIC LUPUS ERYTHEMATOSUS
235
On the other hand, antinuclear antibodies may be related to the extent and
severity of the disease. Both clinically and serologically, patients in Group I
represent a more active and diffuse form of the disease.
The reaction most commonly used to establish the diagnosis of S.L.E. is the
L.E. cell test. This test is reported to be positive in 90 to 100 per cent of
patients with S.L.E.21.25*26
In this study, the patients have been grouped on the
basis of the presence or absence of L.E. cells. If the incidence of L.E. cells in
patients suffering from S.L.E. is over 90 per cent, we should conclude that the
patients in Group I1 were suffering from a disorder other than S.L.E. However, the clinical and serological data reveal a surprising similarity between
the two groups and we may assume that the patients in Group I1 have S.L.E.
as well as those in Group I. If this is correct, it appears that the incidence of L.
E. cells in patients with S.L.E. is not as high as has been thought in the past. If
all 53 patients reported here are grouped together, the incidence of positive
L.E. cell tests is 71.5 per cent. It should be stressed that the L.E. cell tests were
performed in the same laboratory which previously reported L.E. cells in 96
per cent of a series of patients with S.L.E.26Thus, the lower incidence of L.E.
cells reported in the present study cannot be ascribed to the use of special
techniques or the use of special criteria in evaluating the tests. It seems probable, therefore, that the diagnosis of S.L.E. has not previously been made in
many borderline cases unless the L.E. cell tests were positive.
The specificity of the L.E. cell test must also be considered. L. E. cells
have been reported in patients with uncomplicated rheumatoid a r t h r i t i ~ , ~ , ~ ~
systemic scleroderma,B systemic dermatomy~sitis,'~and in hydralazine disease.28,29.30
The absence of L.E. cells in some patients with typical clinical features and
characteristic histological lesions has been explained by assuming that massive
intravital nuclear fixation of circulating antibody has left little or no antibody
available in the circulating blood for in vitro d em~n s t rat i o nHowever,
.~~
if this
were a general rule, the L.E. cell test should be negative in patients with massive and multiple organ involvement. This is not the case in the patients presented here.
The fluorescent antibody t e c h n i q t ~ e ~ land
J ~ *the
~ ~ antiglobulin consumption
method34 have been reported to be the most sensitive of the serological tests
developed in recent years. The sensitivity can be modified by varying the
technical procedure. The fluorescent antibody technique used in this study
gave a low incidence of positive results in random ward patients ( 2 per cent ) .
The incidence of positive tests by this method in all 53 patients was 81 per
cent (Group 1-87 per cent; Group 1 1 - 6 6 per cent), which is higher than the
incidence of positive L.E. cell tests (71.5 per cent) among all patients. The
incidence of positive tests by the fluorescent antibody method was lower
than that reported in S.L.E. by othersllJ2 and is also lower than the incidence
of positive tests reported with the antiglobulin consumption meth0d.3~This
parallels the higher number of patients with negative L.E. cell testa in this
series. The fluorescent antibody method therefore should have great diagnostic
significance. It is fortunately easy to perform. However, this reaction, as well
RS the L.E. cell test, is positive in a relatively large number of pdtients with
236
ROTHE'IELD, PHYTHYON, MC EWEN AND MIESCHER
rheumatoid arthritis (table 5 ) . It can also be positive in patients with
~cleroderma.'~.~~
Of the antinuclear factors detected by the complement fixation method,
anti-DNA antibodies are the most specific for S.L.E. Other reports indicate that
antibodies to DNA have been found only in patients with S.L.E.Qand in occasional patients with rheumatoid arthritis.:j6 Although the specificity of the
anti-DNA reaction is therefore very high, the incidence of positive reactions to
DNA in the 53 patients tested was relatively low (41.5 per cent).
Antibodies to nucleoprotein were found in 55 per cent of all patients tested.
The higher incidence of nucleoprotein reactions generally found in S.L.E.,
compared to reactions to DNA, contrasts with the slightly lower specificity of
the nucleoprotein reaction. This is obvious from the results on control and
rheumatoid arthritis patients as reported here and from findings reported elsewhere of positive nucleoprotein reactions in patients with rheumatoid arthritis,:ii systemic scleroderma,' ~~m systemic dermat~myositis,".'~ and bilinry
cinhosi~.~g
Antibodies to histone were found in very few of the patients reported here.
The specificity of this reaction is low. In a similar study carried out in Switzerland the reaction was positive in a similar percentage of patients suffering
from rheumatoid arthritis?? Indeed, anti-histone antibodies were also found in
some patients with acute bacterial infections.
The overall incidence of positive antinuclear reactions as performed by the
three methods used in this study, i.e., L.E. cell, fluorescent antibody and complement fixation, was 98 per cent. Thus 52 of all 53 patients had a positive reaction by at least one of the above methods. The patient with negative reactions to all tests has been described and discussed above.
On the basis of these data, it appears that the present concept of S.L.E.
may still be too rigid, since it does not take into full consideration the many
milder forms of the disease. The clinical concept of the disease must continue
to be modified as newer data become available. Since the etiology and pathogenesis of S.L.E. are unknown, any rigid definition would be premature and
might actually lead to a limitation of our understanding of the disease,
With the possible exception of hematoxylin bodies, there is no generally accepted pathognomonic histological lesion. Although several lesions are characteristic of the disease, some patients with the unequivocal clinical diagnosis of
S.L.E. fail to show any characteristic lesions at post mortem e~amination.~'!
It
is therefore obvious that if S.L.E. were defined on the basis of pathological
findings, many typical cases would be excluded.
Immune biological reactions have become of great importance in establishing
the diagnosis. The diagnosis of S.L.E. becomes more probable when more antinuclear reactions are positive. On the other hand, repeated negative reactions
obtained by more than one of the sensitive methods to detect antinuclear factors
make the diagnosis exceedingly unlikely.
The problem of the small but important group of patients with rheumatoid
arthritis and positive antinuclear reactions remains to be clarified by long term
clinical and serological studies. Any clear-cut classification of these borderline
cases is premature and arbitrary.
ANTINUCLEAR REACTIONS IN SYSTEMIC LVPUS ERYTHEMATOSUS
237
SUMMARY
1. The clinical and laboratory data on 53 patients with a clinical syndrome
typical of S.L.E. have been analyzed on the basis of the presence or absence of
the L.E. cell phenomenon.
2. Both groups appear to represent one homogeneous disease entity.
3. The importance of the L.E. cell test in establishing the diagnosis of S.L.E.
has been overestimated in the past. The significance of the most important
antinuclear reactions are discussed.
4. The antinuclear antibodies do not reflect the involvement of particular
organs. Their incidence is more frequent in the more severe forms of S.L.E.
and in the active state of the disease process.
5. The diagnosis of S.L.E. is discussed with regard to the use of antinuclear
reactions as a diagnostic tool.
ACKNOWLEDGMENTS
We wish to express our gratitude to Mr. Martin Tanner for performing the tests for
rheumatoid factor and to Dr. N. S. Cooper for reviewing the pathological material obtained
at autopsy.
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ANTINUCLEAR REACTIONS IN SYSTEMIC LUPUS ERYTHEMATOSUS
Naomi F. Rothfield, M.D., Assistcln.t in Medicine, New York
University School of Medicine, New York, N . Y.
James Phythyon, M.D., Fellow, The National Foundation,
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Currier McEwen, .M.Do,Professor of Medicine and Chairman,
Rheumatic Diseases Study Group, New York Uniuersity School
of Medicine, New York, A’. Y.
Peter Miescher, M.D., Professor of Medicine, New York Uniuersity School of Medicine, New York, N . Y.
239
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