L u p u s
E r y t h e m a t o s u s
A n t i n u c l e a r
Cell
Factor
P r e p a r a t i o n -
I n c o n g r u i t y
A Review of Diagnostic T e s t s for Systemic L u p u s E r y t h e m a t o s u s STEPHEN R. ROLLER, M.D., CHARLES L . J O H N S T O N , J R . , M.D., CHARLES W. MONCURE,
M.D.,
AND MARION V. WALLER, P H . D .
From the Division of Immunology and Connective Tissue Diseases, Department of Medicine, and the Depa Pathology, Medical College of Virginia, Health Sciences Division, Virginia Commonwealth Univers Richmond, Virginia 23298 ABSTRACT
" T H E indirect immunofluorescent technic for antinuclear antibodies, when 'properly performed,' would be positive in nearly every case where the LE cell phenomenon is positive." 1 This widely accepted statement has led to the discontinuance in some laboratories of the lupus erythematosus (LE) cell preparation and its replacement by the antinuclear factor (ANF) test as a screening test for the diagnosis or exclusion of systemic lupus erythematosus. There are, however, references to
the occurrence of a positive LE cell preparation with a negative ANF test. 933 Clinical information concerning these patients remains unpublished. Since 1971, we have identified 40 patients who had this phenomenon. Such patients continue to be referred to us at the rate of approximately one a month. We are fortunate to have had available the acute sera of 20 such patients. It is our aim to analyze and explain this serologic paradox,
Materials and Methods Received July 10, 1975; received revised manuscript October 20, 1975; accepted for publican , • , n . , ,• tion October 20, 1975. Patient Population Supported in part by Charles VV. Thomas Arthritis ™. ,-,.. . . . . .. .. l he Fund. This is publication number 90 from the Clinical Laboratories ot the Medical Charles w. Thomas Arthritis Fund. College of Virginia from 1971 through Address reprint requests to Dr. Roller: Department ,C.IA C J . i o onrv il i1974 of Medicine^ Kenner Army Hospital, Fort Lee, performed approximately 2,200 LL Virginia 23801. cell preparations per year. All those intcr495
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Roller, Stephen R., Johnston, Charles L., Moncure, Charles W., and Waller, Marion V.: Lupus erythematosus cell preparation-antinuclear factor incongruity. A review of diagnostic tests for systemic lupus erythematosus. Am J Clin Pathol 66: 4 9 5 - 5 0 5 , 1976. T h e cases of 20 patients, each of whom had a positive lupus erythematosus cell preparation and a negative antinuclear factor test, are presented. T h e concept of a false-positive lupus erythematosus preparation is suggested. Five common mechanisms causing a false-negative antinuclear factor test are discussed and evaluated. Clinical material from the 20 patients is described and pitfalls in diagnosing systemic lupus erythematosus are reviewed. (Rey words: Antinuclear factor; Lupus erythematosus cell preparation; Pseudo-LE cell; Systemic lupus erythematosus.)
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preted as positive were saved. The records of patients whose lest were positive were examined to determine whether an ANF test had also been performed and, if so, the result was recorded. Forty patients were identified as having a negative ANF test and a LE cell preparation interpreted as positive. Sera of 20 of the patient were supplied by the rheumatoid arthritis serology laboratory. These sera were harvested within 48 hours, usually at the same time the ANF test (neg.) and LE cell preparation (pos.) were performed.
LE Cell Preparation. Five milliliters of venous blood were placed in a screwcapped vial containing four to six, 3-mm glass beads and shaken vigorously for 5 minutes. The specimen was incubated for two hours and passed through a 40-mesh copper screen (425 (im) to remove fibrin clots and beads. T h e strainings were placed in a Seditube* and centrifuged for 10 minutes at 325g. The buffy coat was collected and four smears were prepared. Dried smears were fixed with methyl alcohol and stained with Wright's stain. Each slide was viewed for 10-15 minutes. When a slide had two or more classic LE cells, or when two slides each contained one classic LE cell, the result was considered positive. ANF Test, Fresh-frozen 6-/n,m sections of mouse liver were air-dried. Sera at a screening dilution of 1:20 were placed on the sections and incubated for 30 minutes at room temperature. T h e slides were then rinsed and washed with gentle agitation in two changes of phosphate-buffered saline solution (PBS) for a total of 15 minutes. T h e sections were then covered with an appropriate dilution of fluorescein-conjugated goat anti-human immunoglobulint * Becton-Dickinson. Rutherford. New Jersey. t Meloy #C-301. dilution determined by checkerboard titration.
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and incubated at room temperature for 30 minutes. The slides were again rinsed and washed in three changes of PBS for a total of two hours. The sections were covered with buffered glycerol and coverslips and examined with a Zeiss fluorescence microscope. Special Studies LE Verification. All slides were reviewed by one technician and one of the authors (S.K.) in a blind protocol, with six negative slides interspersed among the test slides. Qualitative LE Readings. All slides were reviewed by one of the authors (S.K.) and one technician in a blind protocol LE cell preparations of nine patients with known systemic lupus erythematosus (SLE) were interspersed among the test slides. T h e numbers of classic LE cells, pseudo-LE cells, and tart cells were counted. Each slide was viewed for 10 minutes under oil or until 30 cells of these three types had been counted. ANF Fixation. The ANF test was repeated as described above, using fixed and unfixed nuclear substrate. Each serum was tested at a dilution of 1:20. Fixation was accomplished by immersion of tissue slides in acetone for 10 minutes at room temperature. ANF Verification. T h e sera were also tested in the laboratory of Dr. Naomi Rothfield.30 Conjugate Verification. T h e ANF test was repeated using a conjugate known to contain antibody activity against all immunoglobulin classes and light chains.:]: Granulocyte Antigens. Ten milliliters of whole blood were drawn into a heparinized tube containing 25 units of heparin/ml and gently mixed. One milliliter of Plasmagel§ was added and mixed. T h e specimen was then centrifuged for 8 minutes tMeloy #C-201, dilution 1:10. § Roger Bellon Laboratories, Neuilly, France.
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Routine Laboratory Procedures
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Varying Fixation. Various fixatives were used to fix leukocyte smears prior to serum application. These included ethyl alcohol (cold, 70%, for 10 minutes), ethyl alcoholether (50/50 dilution, cold, for 10 minutes), and acetone (cold and room temperature for 10 minutes). DNA Binding. DNA binding was done in the laboratory of Dr. Naomi Rothfield. 20 Results Both reviewers verified the laboratory's readings of all slides (26 of 26). T h e phagocytic cell was often likely to be a monocyte rather than a polymorphonuclear leukocyte. Table 1 summarizes the clinical diagnoses of the 20 patients. Six patients had rheumatoid arthritis, three had major d r u g reactions, two had tendonitis, and nine had other conditions. Table 2 summarizes the organ-system involvement and pertinent laboratory tests of the 20 patients and tabulates the provisional criteria 7 for systemic lupus erythematosus each fulfilled. Three patients (9, 14, and 17) each satisfied four or more criteria for systemic lupus erythematosus. Patient 9 had had dis-
coid lupus in the past, and had lymphopenia (leukocyte count 3,500), a butterfly rash, and arthralgias and pain on motion of both ankles. Patient 14 had degenerative joint disease of both knees, longstanding alopecia on one side of her head, and facial photosensitivity. Patient 17 had intermittent pleuritis, one episode of nasal ulceration, xerostomia, and Raynaud's phenomenon. Most of the other patients had multiple organ-system involvement, but their disease did not fulfill the rigid criteria needed for the diagnosis of systemic lupus erythematosus. These patients had involvement of the same organs commonly involved in systemic lupus erythematosus, but signs, symptoms, and laboratory values were not diagnostic of systemic lupus erythematosus and did not satisfy the American Rheumatism Association (ARA) criteria (Table 3). For example, renal involvement, to satisfy an ARA criterion, requires casts or proteinuria with values in excess of 3.5 g per day. Pyuria, hematuria, decreased creatinine clearance, or lesser proteinuria may occur in SLE, but do not satisfy the provisional ARA criteria. Specimens from 19 of 20 patients each had at least one tart cell or pseudo-LE cell. Eighteen of the 19 had pseudo-LE cells and tart cells equaling or outnumbering classic LE cells (Table 1 and Fig. 1). No patient of the nine with known SLE had a ratio of classic LE cells to pseudoLE cells of less than 1.0. Three patients (5, 6, and 18) had higher titers of ANF when the nuclear substrate was fixed prior to application of test serum (Table 1). One of these patients (18) had a positive ANF test result of 1:20 (unfixed) two months before her LE cell preparation was positive. However, by the time the LE cell preparation was performed, her ANF test result had become negative, and she had never received treatment. Seven patients (3, 4, 5, 6, 9, 12, 17) (35%)
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at 160 X g. T h e supernatant was removed and centrifuged at 475 X g for 6 minutes. The cells in the pellet were washed in PBS three times and centrifuged at 200 x g for 6 minutes after each wash. One drop of the final suspension was placed on a clean glass slide using a capillary tube and spread with a coverslip. The slides were air-dried and either used immediately or stored at 4 C for less than a week. Defibrinated leukocytes were prepared by vigorously shaking 10 ml of fresh whole blood for 10 minutes with glass beads and adding 1 ml Plasmagel, and treated as described above. Damaged leukocytes were created by allowing the final suspension of heparinized leukocytes to incubate at 4 C overnight. The cells were fixed (see below). Serum was then placed on the substrate and treated as in the ANF test described above.
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Table 1. Clinical Diagnoses and Experimental Results in Cases of 20 Patients with Positive LE Cell Preparations and Negative ANF Tests
Case
Diagnosis
Comments
Classic LE Pseudo LE and Tart Cells
Gain in Titer with Tissue Fixation
ANA in Low Titer
GS-ANF
Perivascular Fluorescence Titer 1:8
Probable rheumatoid arthritis
Seropositive
3/26
X
2
Probable rheumatoid arthritis
Seropositive
6/24
X
3
Definite rheumatoid arthritis
Seropositive
3/4
X
4
Definite rheumatoid arthritis
Seropositive
1/11
X
5
Definite rheumatoid arthritis
Seropositive
1/3
X
X
6
Definite rheumatoid arthritis
Seropositive
17/13
X
X
7
Drug reaction
Sulfamethoxazole
1/29
8
Drug reaction
9
Drug reaction
Chlorpropamide Sulfamethoxazole phenoxymethyl PCN
3/8 2/2
10
Tendonitis
6/11
11
Tendonitis
3/13
12
Avascular necrosis
13
Hypersensitivity pneumonitis Degenerative joint disease
14 15
Secondary to steroids ? Antigen Discharge diagnosis of SLE
X
X
X
X X
3/27
X
1/7
Discharge diagnosis of SLE
1/0
Discharge diagnosis of SLE
1/1
18
CVA with monoarticular arthritis
Discharge diagnosis of SLE
1/29
19
Skin rash
Discharge diagnosis of SLE
1/2
20
Magnesium deficiency
Alcoholic myopathy
3/27
17
X
1/6
Juvenile rheumatoid arthritis Recurrent juvenile rheumatoid arthritis in adult Sjogren's syndrome
16
X
X
1/29
X X
X
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1
Nonspecific abnormalities Deforming arthritis Dermatologic Renal Central nervous system Hematologic Per cent DNA binding
TOTAL CRITERIA
Provisional ARA criteria for SLE Arthritis without deformity Central nervous system Serositis Raynaud's phenomenon Mucous membrane ulcers Photosensitivity Facial erythema Discoid lupus Alopecia Hematologic Proteinuria Cellular casts False-positive serologic test for syphilis LE cells
X
12
X
12
X
X
8
X
X
3
X
X X 1 2
1 2
X 8
X
X 1 2
X
4
X
6
6
X X X X 10
X
X X 1 2
5
12
X X X
X 2
X
7
11
X
X 1
8
16
X X
X 5
X
X X
X
9
3
X
10
1
17
X
11
Patient
X 13
X
X 1 1
12
18
X 2
X
13
0
X
X
12
X
X
15
X 4
X
X X
14
X
33
X 30
X 4
X X X
17
X
X 1 2
X
16
9
X
X
X 2
X
18
8
X
X
19
20
6
X X
X 1 1
Table 2. Presence or Absence of Nonspecific Abnormalities and Signs, Symptoms, and Laboratory Values Composing the ARA Criteria for SLE in 20 Patients with Positive LE Cell Preparations and Negative ANF Tests
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to
o c •
z
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using leukocytes as a nuclear substrate. In both cases, these patterns were found with undiluted serum only. Varying the method of preparation and fixation of substrate did not affect the generally negative results for granulocyte-specific antinuclear factors (Fig. 2). Using defibrinated leukocytes did not increase sensitivity. Both patients were leukopenic. No patient had DNA binding within the range seen in active systemic lupus erythematosus. Discussion LE Preparation The positive LE cell test as currently defined necessitates the presence of at least two classic LE cells on the slide or one classic LE cell on each of two slides. Formation of the LE cell is a multi-stage process that requires four distinct elements: (1) nuclear substrate; (2) LE factor; (3) phagocytic cell; (4) complement. Most laboratories use a direct LE cell test using autologus
Table 3. Detailed Analysis of Nonspecific Organ-system Involvement in 20 Patients with Positive LE Cell Preparations and Negative ANF Tests System
Patient
Abnormality
CNS (3)
6 7 18
Mild depression Lethargy and coma Cerebrovascular accident age 34 years due to small-vessel occlusion
Hematologic (4)
4 6 12 16
Positive Coombs' test, not anemic, had previously received blood transfusion Unexplained anemia Unexplained anemia Unexplained anemia
Dermatologic (3)
7 8 9
Renal (10)
2 3 6 7 9 15 16 17 19 20
Total-body erythema Erythematous maculopapular and vesicular lesions on the trunk Longstanding frontal alopecia Trace proteinuria Trace proteinuria, pyuria Trace proteinuria, hematuria Pyuria, proteinuria, hematuria, decreased creatinine clearance Pyuria, proteinuria Pyuria Proteinuria (1 g), decreased creatinine clearance Pyuria, hematuria Proteinuria (430 mg), pyuria Pyuria
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had low titers of ANF that would ordinarily be considered negative by the laboratory. One patient (3) had a low titer of ANF at the time of the study, but within 6 months, the ANF test result became positive at a titer of 1:100. Specimens from nine patients had fluorescent patterns on the mouse liver that were not nuclear in position. T h e patterns were perivascular and sinusoidal (Table 1). T h e results of ANF testing were verified in the reference laboratory. 30 This laboratory routinely fixes mouse liver sections. T h e results were almost identical to the results obtained at the Medical College of Virginia. Three patients had titers of 1:16 or more. T h e same seven patients who had low titers in our laboratory and two additional patients had low titers of ANF using undiluted sera, and specimens from the same nine patients showed non-nuclear fluorescence. Specimens from two patients (7 and 13) had positive diffuse fluorescent patterns
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LE-ANF INCONGRUITY ©
o=>>-#->!50
I 50
Clossic LE cells Pseudo LE cells plus tart cells
IOO
0 75 0 50 0 25
000
A •v Systemic Patients in Study Lupus (20) Erythematosus (9)
fl) only one abnormal cell seen on this slide
FIG. 1. Ratios of classic LE cells to pseudo-LE cells plus tart cells in LE preparations, 20 patients with the LE cell preparation-ANF test incongruity and nine patients with known SLE.
10
Substrate / Fixation / Dilution
15
20 _i
Liver / unfixed / 1: 2 0 Liver /acetone / 1:20
Fie. 2. Results of special ANF tests of 20 patients with the LE cell WBC /cold ETOH /(undiluted) preparation-ANF test incongruity. Conditions of the substrate, fixation technic, and dilution of serum are WBC /cold ETOH-ether /(undiluted) shown on the left. The number of patients tested with each technic and the number of positive tests Aged WBC /cold ETOH /(undiluted) (shaded area) are denoted by bars. WBC/acetone /(undiluted)
positive negative
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cells as the source of nuclear material. However, when the patient is leukopenic, homologous or even heterologous cells may be used in an indirect test. The nuclear substrate from certain animal sources may, in fact, be more sensitive to the action of LE factor than human substrate. 5 It has also been shown that either granulocytes 19 or lymphocytes 25 lend themselves to use as nuclear substrate in the LE cell test. More important than the source of the nuclear material is its viability. The LE factor will react only with nuclei 23 and, therefore, the factor must penetrate the cytoplasmic membrane. Diffusion of the LE factor across the cytoplasmic membrane can be facilitated by damaging that membrane. This can be effected by: vigorous shaking with glass beads; crushing and straining a clot through a fine-mesh sieve; drying the cells; freezing and thawing the cells; or chemical injury. The factor does not affect living cells,27 and hence it is rare to find an LE cell in vivo except in those special cases where numerous damaged cells are in a closed space and cannot
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Other variables also affect the LE cell preparation. High concentrations of heparin will inhibit the LE phenomenon. Additionally, an adequate incubation time is required. The mechanism of the LE phenomenon was studied in detail by Robineaux. 29 On adding the lupus factor to substrate nuclei, changes are evident almost instantly. As the
nuclei are homogenizing, the cytoplasmic granules become perinuclear. Finally, the cytoplasmic membrane ruptures, and only then do other polymorphonuclear cells become attracted to the affected nucleus. T h e nucleus must be in contact with the environment for this process to occur. T h e phagocytic cell will engulf the free or exposed nucleus, leaving the cytoplasm unincorporated. Tart cells and pseudo-LE cells may resemble LE cells. Tart cells, described at the same time the LE cell was described, 16 were named after the patient in whom they were found. T h e mechanism of their formation is not as well worked out as that of the LE cell. Kurnick 18 has observed that pseudo-LE cells can be formed as a result of an entire cell's being phagocytized. As intracytoplasmic breakdown occurs, the nucleus of the inclusion cell becomes more homogeneous. The reticular pattern is lost and a basophilic ring appears around the border of the inclusion. Rarely, all staining differences between the LE nucleus and the pseudo-LE nucleus may be lost, either by the above-described mechanism or by another, unknown, mechanism, and the cell may mimic a classic LE cell.15 Tart and pseudo-LE cells have been produced in the presence of leukoagglutinins. 12 Indirectly supporting this concept is the fact that pseudo-LE cells are found in patients with drug reactions, 22 which may also be associated with antibodies to blood components. Forty per cent of patients who have SLE may have luekoagglutinins present. 8,10 T h e problem with interpreting data regarding leukoagglutinins lies in the lack of standardized procedures and the lack of differentiation between autoantibodies and isoantibodies. It is also difficult to distinguish between antibodies directed against surface antigens and immune complexes nonspecifically bound to the cell membrane. T h e presence of factors directed against lymphocytes has been well-established. 24
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be easily cleared. Such spaces include joint, 17 pericardial, 31 and pleural 32 cavities. T h e LE factor is usually a 7S IgG. Other anti-deoxynucleoprotein antibodies that are not IgG but instead IgM have been found. These, however, have less tendency to form LE cells because of their low affinity for complement. 4 When the LE factor combines with the nuclear substrate, the chromatin pattern of the nucleus is gradually lost, and eventually the nucleus becomes homogeneous. The homogeneous mass is then exposed to the environment, either partially through a slight disruption of the cytoplasmic membrane, or totally by extrusion of the mass. Once feee, it is called a "hematoxylin body." In order for the hematoxylin body to be engulfed by another phagocyte a serum factor must be present. This factor is thought to be complement because it can be removed by heating the LE serum to 56 C for 30 minutes and because it can be deactivated by anti-C3. Exacdy how much complement is necessary for the completion of the LE test is unknown, but it is thought to be very little.13 Without complement, many hematoxylin bodies are found. Hematoxylin bodies by themselves are not diagnostic, but they are often seen in conjunction with LE cells. On the other hand, in the presence of complement, the hematoxylin body can be phagocytized by polymorphonuclear leukocytes (occasionally either a less mature cell of the myelocytic series or a monocyte) to form an LE cell. T h e phagocytizing cell will become swollen and its nucleus will become displaced into a peripheral position.
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Antinuclear Factor Having established the classic LE cells were present, and setting aside the concept of a false-positive LE cell preparation for the moment, the issue of a falsenegative ANF test result was raised. It seemed appropriate to review the mechanisms of the ANF test and the possible ways by which false-negative results can occur. T h e positive ANF test is a threelayered test: Layer 1, nuclear substrate;
layer 2, patient's serum containing antinuclear factors; layer 3, fluorescein-conjugated anti-human immunoglobulin, monovalent or polyvalent. Tissues that have been utilized to supply nuclear material include kidney (mouse and rat), liver (mouse and rat), tumor imprints, buccal mucosa, spleen imprints, tissue culture cells, chick erythrocytes, human leukocytes, and calf thymus. Once a tissue section is placed on the slide, the section may or may not be fixed. Several mild fixatives are in use, acetone and ethyl alcohol being the most common. It had been shown by Beck2 and later by Claymaet and Nakamura 6 that without fixation, some soluble nuclear antigens would be lost. We had three patients whose titers of ANF increased as a result of fixing. Two had finely speckled patterns and one had a diffuse pattern. There is no standard method for ANF determinations. One is clearly needed, and we feel that fixation should be a part of any standard procedure. Layer 2 is the patient's antinuclear factors). There are three ways in which the patient may have antinuclear factor but a negative antinuclear factor test result: (1) ANF present but entirely bound to circulating antigen; (2) ANF present and unbound, but titer too low to be discovered at screening dilution; (3) ANF present but organspecific, i.e., it can react with nuclei of leukocytes, giving a positive LE cell preparation, and not react with other tissue antigens, giving a negative ANF test result. It has been shown that immune complexes, when present, may produce falsenegative ANF test results. 3 In such a case, when the serum undergoes acid dissociation of the immune complexes, the ANF test result becomes positive. It is unlikely that such a mechanism was operative in our patients. The LE cell preparations were positive, implying the presence of circulating unbound antinuclear factors. In addition, one would expect that the presence of the immune complexes would
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Whether these factors are a primary or secondary phenomenon is still under investigation. The theory presented regarding the formation of the pseudo-LE cells has been thought by some to be oversimplified. 21 Several investigators 21 have reported that tart and pseudo-LE cells may precede the development of the classic LE phenomenon. We have seen free cell nuclei that resemble the inclusion of the pseudo-LE cell in LE cell preparation slides. It must be remembered that in evaluating studies where the LE cell is of critical importance, stringent criteria for the identification of the LE cell must be strictly observed. There have been several reports in the literature where the LE cell appeared to have been a pseudo-LE cell. After careful review of the 20 cases reported in this paper, the predominant cell was found to be the pseudo-LE cell or tart cell in specimens from 18 of the 20 patients, whereas the opposite was the case in all nine SLE specimens. We believe that LE cell preparation in which pseudo-LE cells and tart cells greatly outnumber classic LE cells cannot be used to verify the diagnosis of SLE. Consequently, we recommend that the characteristics of the entire slide should be examined. One or two classic LE cells do not, by themselves, in a milieu of tart and pseudo-LE cells, have diagnostic significance, because they may have attained their morphology by the leukoagglutination pathway.
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Finally, the fluorescent conjugate may be responsible for a falsely negative result. If, for example, the ANF is an IgM, as it may be in 4% of patients, 14 and the conjugate is monovalent for IgG, no fluorescence will be evident. Special polyvalent conjugate did not increase the yield of positive ANF tests. An interesting finding was the identification of nine patients whose specimens showed a perivascular sinusoidal pattern at a dilution of 1:8 or more. This pattern is similar to that seen with antivascular antibody 26,34 and antireticulin antibody. 28 T h e diagnoses of our 20 patients varied. Rheumatoid arthritis seemed to predomi-
66
nate, but this could be due to the fact that the sera had been obtained from the rheumatoid arthritis serology laboratory. No clear clinical pattern was evident. All the patients, however, seemed to have signs, symptoms, and laboratory findings that suggested systemic disease. The findings were similar, but not identical, to those that the ARA enumerates for systemic lupus erythematosus (Table 2). Due to inappropriate application of the ARA criteria, five patients initially had been incorrectly diagnosed as having SLE. Summary The cases of 20 patients who had positive LE cell preparations and negative ANF test results were studied. The reasons for this serologic paradox are discussed. Three patients had false-negative ANF test results because the sections were not fixed and the soluble antigens were lost during the performance of the test. One patient had a titer of ANF that was below the screening level but was subsequently elevated several months later. The problem of a falsepositive LE cell preparation is discussed. Other mechanisms are discussed and rejected as reasons for the serologic abnormalities. No clearly identifiable syndrome emerges from clinical analysis of these 20 cases. The following conclusions can be drawn, based on our experience: (1) T h e nuclear substrate in the ANF test should be fixed. (2) Although we do not recommend full titering of the ANF for every patient, there will be an occasional case in which it would be helpful. (3) When classic LE cells are greatly outnumbered by pseudo-LE cells or tart cells, or both, a positive LE cell preparation has limited diagnostic value, and should not be used to verify a diagnosis of SLE or to fulfill the provisional ARA criteria. (4) With the limitations and mechanisms of the ANF test and LE cell preparation understood, it is justifiable to use the ANF test as a screening test for connective-tissue diseases. However, when because of technical considerations the ANF test is not as reliable as it might be, the LE cell prepara-
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bind complement and hence lower its serum concentration. T h e C3's of the patients for whom this information was available were normal. In addition, DNA binding was in the normal range for all patients (Table 2). One patient had a titer of ANF below the dilution screening point and later developed a more than fourfold increase in titer. When the clinical or laboratory picture warrants it, we believe the ANF test should be done at lower dilutions. T h e existence of granulocyte-specific ANF has been known since 1964.11 These factors react preferentially with the nuclei of granulocytes. They are measured using leukocytes as a nuclear source, by finding a higher titer present against these leukocytes than against human thyroid cell nuclei. Granulocyte-specific ANF have been found in association with rheumatoid arthritis, especially with leukopenia and/or Felty's syndrome. In general, we have found that human leukocytes in smears are less sensitive than mouse liver as a nuclear antigen source. We found two patients who had low titers of granulocytespecific antinuclear factors, but positive tests were obtained using different fixative technics and were of low titer, the results being weakly positive in undiluted sera. These two patients both had leukopenia. Varying the fixative and preparation technic did not increase the yield of granulocyte-specific ANF.
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A morphological study. South Med J 49:560566, 1956 16. Hargraves MM, Richmond H, Morton R: Presentation of two bone marrow elements: The "tart" cell and "L.E." cell. Proc Staff Mayo Clin 23:25-28, 1948 17. Hunder GG, Pierre RV: In vivo LE cell formaAcknoxukdgments. Mrs. Nellie Curry, Mrs. Loretta tion in synovial fluid. Arthritis Rheum 13:448Albert, and Mrs. Carolyn Jones provided technical 450, 1970 assistance. Dr. Shaun Ruddy and Dr. Keith Whaley offered suggestions and encouragement. Help and 18. Kurnick NB: The pseudo-LE cell phenomenon with report of a case. Blood 12:382-390, guidance were provided by Dr. Naomi Rothfield of 1957 the University of Connecticut School of Medicine. 19. Larson RA, Boyum A: Development of an indirect LE factor test. Acta Med Scand 543: References 21S-29S, 1972 1. Barnett EV, Rothfield N: The present status of 20. Luciano A, Rothfield NF: Pattern of nuclear antinuclear antibody serology. Arthritis Rheum fluorescence and DNA-binding activity. 12:543-544, 1969 Ann Rheum Dis 32:337-341, 1973 2. Beck JS: Variations in the morphological patterns 21. Marmont A: Nucleolytic phagocytosis (L.E. cell of "autoimmune" nuclearfluorescence.Lancet phenomenon) in systemic lupus erythematosus, 1:1203-1205, 1961 rheumatoid arthritis, and systemic sclerosis, 3. Blomjous FJEM, Feltkamp-Vroom TM: Hidden First International Symposium on Immunoantinuclear antibodies in seronegative systemic pathology. Edited by Grabar P, Miescher P. lupus erythematosus patients an in NZB and Basel, Benno Schwabe and Co. Verlag, 1958, (NZB X NZW) Fl mice. Eur J Immunol pp 479-502 1:396-398, 1971 22. Meyler L: Phagocytosis in drug allergy. Acta 4. Blondin C, McDuffie FC: Role of IgG and IgM Med Scand 150:33-38, 1954 antinuclear antibodies in formation of lupus 23. Mieschner P, Fauconnet M: L'absorption du erythematosus cells and extracellular material. facteur "L.E." par des noyaux cellulaires Arthritis Rheum 13:786-797, 1970 isoles. Experientia 10:252-254, 1954 5. Carrera AE, Reid MV, Kurnick NB: Differences 24. Mittal KK, Rossen RD, Sharp JT, et al: Lymphoin susceptibility of polymorphonuclear leukocyte cytotoxic antibodies in systemic lupus cytes from several species to alteration by erythematosus Nature (Lond) 225:1255systemic lupus serum: Application to a more 1256, 1970 sensitive L.E. phenomenon. Blood 9:1165- 25. Moyer JB, Fisher GS: Experimental production 1171, 1954 of "L.E." cells. Am J Clin Pathol 20:1011-1015, 6. Cleymaet JE, Nakmura RM: Indirect immuno1950 fluorescent antinuclear antibody tests: Com- 26. Person DA, Leatherwood CM, Sharp JT: Antiparison of sensitivity and specificity of different vascular antibody. Ann Rheum Dis 33:371substrates. Am J Clin Pathol 58:388-393, 375, 1974 1972 27. Rapp F: Localization of antinuclear factors from 7. Cohen AS, Reynolds WE, Franklin EC, et al: lupus erythematosus sera in tissue culture. Preliminary criteria for the classification of J Immunol 88:732-740, 1962 systemic lupus erythematosus. Bull Rheum Dis 28. Rizzetto M, Doniach D: Types of "reticulin" 21:643-648, 1971 antibodies detected in human sera by immuno8. Dausset J, Colombani J, Colombani M: Study of fluorescence. J Clin Pathol 26:841-851, 1973 leukopenias and thrombocytopenias by the 29. Robineaux R: Study of LE cell formation by direct antiglobulin consumption test on leukophase contrast microcinematography, Mechcytes and/or platelets. Blood 18:672-690, 1961 anisms of Hypersensitivity. Edited by Saffer 9. Dubois EL: Lupus Erythematosus. Los. Angeles, JH, LoGrippo GA, Chase MW. Boston, California, University of Southern California Little, Brown and Company, 1959, pp 371Press, 1974, p 487 395 Engelfriet CP, Van Loghem JJ: Studies on leuco10 NF, Fragione B, Franklin EC: Slowly cytes iso- and auto-antibodies. Br J Haematol 30. Rothfield sedimenting mercaptoethanol-resistant anti7:223-238, 1961 nuclear factors related antigenically to "M" 11 Faber V, Elling P, Norup G, et al: An antiimmunoglobulins (ym-globulin) in patients nuclear factor specific for leucocytes. Lancet 2: with systemic lupus erythematosus. J Clin 344-345, 1964 Invest 44:62-72, 1965 12 Finch SC, Ross JF, Ebaugh FG: Immunologic mechanism of leukocyte abnormalities. J Lab 31. Seaman AJ, Christeron JW: Demonstration of L.E. cells in pericardial fluid. JAMA 149:145Clin Med 42:555-569, 1953 147, 1952 Golden HE, McDuffie FC: Role of lupus erythe13 matosus factor and accessory serum factors in 32. Ibid: See footnote Grondahl R production of extracellular nuclear material. 33. Souhami RL, Homma M, Gumpel JM, et al: The absence of antinuclear factors (by immunoAnn Intern Med 67:780-790, 1967 fluorescence) in certain patients with abnormal 14 Gonzalez EN, Rothfield NF: Immunoglobulin L.E. cell tests. Arthritis Rheum 8:470, 1965 class and pattern of nuclear fluorescence in systemic lupus erythematosus. N Engl J Med 34. Tan EM, Pearson CM: Rheumatic disease sera 274:1333-1338. 1966 reactive with capillaries in the mouse kidney. 15. Gouldin EN, Diggs LW: L.E. and L.E.-like cells: Arthritis Rheum 15:23-28, 1972 tion should be performed in addition. (5) T h e ARA criteria, when used to support the diagnosis of systemic lupus erythematosus, should be employed with precision.
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E r y t h e m a t o s u s
A n t i n u c l e a r
Cell
Factor
P r e p a r a t i o n -
I n c o n g r u i t y
A Review of Diagnostic T e s t s for Systemic L u p u s E r y t h e m a t o s u s STEPHEN R. ROLLER, M.D., CHARLES L . J O H N S T O N , J R . , M.D., CHARLES W. MONCURE,
M.D.,
AND MARION V. WALLER, P H . D .
From the Division of Immunology and Connective Tissue Diseases, Department of Medicine, and the Depa Pathology, Medical College of Virginia, Health Sciences Division, Virginia Commonwealth Univers Richmond, Virginia 23298 ABSTRACT
" T H E indirect immunofluorescent technic for antinuclear antibodies, when 'properly performed,' would be positive in nearly every case where the LE cell phenomenon is positive." 1 This widely accepted statement has led to the discontinuance in some laboratories of the lupus erythematosus (LE) cell preparation and its replacement by the antinuclear factor (ANF) test as a screening test for the diagnosis or exclusion of systemic lupus erythematosus. There are, however, references to
the occurrence of a positive LE cell preparation with a negative ANF test. 933 Clinical information concerning these patients remains unpublished. Since 1971, we have identified 40 patients who had this phenomenon. Such patients continue to be referred to us at the rate of approximately one a month. We are fortunate to have had available the acute sera of 20 such patients. It is our aim to analyze and explain this serologic paradox,
Materials and Methods Received July 10, 1975; received revised manuscript October 20, 1975; accepted for publican , • , n . , ,• tion October 20, 1975. Patient Population Supported in part by Charles VV. Thomas Arthritis ™. ,-,.. . . . . .. .. l he Fund. This is publication number 90 from the Clinical Laboratories ot the Medical Charles w. Thomas Arthritis Fund. College of Virginia from 1971 through Address reprint requests to Dr. Roller: Department ,C.IA C J . i o onrv il i1974 of Medicine^ Kenner Army Hospital, Fort Lee, performed approximately 2,200 LL Virginia 23801. cell preparations per year. All those intcr495
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Roller, Stephen R., Johnston, Charles L., Moncure, Charles W., and Waller, Marion V.: Lupus erythematosus cell preparation-antinuclear factor incongruity. A review of diagnostic tests for systemic lupus erythematosus. Am J Clin Pathol 66: 4 9 5 - 5 0 5 , 1976. T h e cases of 20 patients, each of whom had a positive lupus erythematosus cell preparation and a negative antinuclear factor test, are presented. T h e concept of a false-positive lupus erythematosus preparation is suggested. Five common mechanisms causing a false-negative antinuclear factor test are discussed and evaluated. Clinical material from the 20 patients is described and pitfalls in diagnosing systemic lupus erythematosus are reviewed. (Rey words: Antinuclear factor; Lupus erythematosus cell preparation; Pseudo-LE cell; Systemic lupus erythematosus.)
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preted as positive were saved. The records of patients whose lest were positive were examined to determine whether an ANF test had also been performed and, if so, the result was recorded. Forty patients were identified as having a negative ANF test and a LE cell preparation interpreted as positive. Sera of 20 of the patient were supplied by the rheumatoid arthritis serology laboratory. These sera were harvested within 48 hours, usually at the same time the ANF test (neg.) and LE cell preparation (pos.) were performed.
LE Cell Preparation. Five milliliters of venous blood were placed in a screwcapped vial containing four to six, 3-mm glass beads and shaken vigorously for 5 minutes. The specimen was incubated for two hours and passed through a 40-mesh copper screen (425 (im) to remove fibrin clots and beads. T h e strainings were placed in a Seditube* and centrifuged for 10 minutes at 325g. The buffy coat was collected and four smears were prepared. Dried smears were fixed with methyl alcohol and stained with Wright's stain. Each slide was viewed for 10-15 minutes. When a slide had two or more classic LE cells, or when two slides each contained one classic LE cell, the result was considered positive. ANF Test, Fresh-frozen 6-/n,m sections of mouse liver were air-dried. Sera at a screening dilution of 1:20 were placed on the sections and incubated for 30 minutes at room temperature. T h e slides were then rinsed and washed with gentle agitation in two changes of phosphate-buffered saline solution (PBS) for a total of 15 minutes. T h e sections were then covered with an appropriate dilution of fluorescein-conjugated goat anti-human immunoglobulint * Becton-Dickinson. Rutherford. New Jersey. t Meloy #C-301. dilution determined by checkerboard titration.
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and incubated at room temperature for 30 minutes. The slides were again rinsed and washed in three changes of PBS for a total of two hours. The sections were covered with buffered glycerol and coverslips and examined with a Zeiss fluorescence microscope. Special Studies LE Verification. All slides were reviewed by one technician and one of the authors (S.K.) in a blind protocol, with six negative slides interspersed among the test slides. Qualitative LE Readings. All slides were reviewed by one of the authors (S.K.) and one technician in a blind protocol LE cell preparations of nine patients with known systemic lupus erythematosus (SLE) were interspersed among the test slides. T h e numbers of classic LE cells, pseudo-LE cells, and tart cells were counted. Each slide was viewed for 10 minutes under oil or until 30 cells of these three types had been counted. ANF Fixation. The ANF test was repeated as described above, using fixed and unfixed nuclear substrate. Each serum was tested at a dilution of 1:20. Fixation was accomplished by immersion of tissue slides in acetone for 10 minutes at room temperature. ANF Verification. T h e sera were also tested in the laboratory of Dr. Naomi Rothfield.30 Conjugate Verification. T h e ANF test was repeated using a conjugate known to contain antibody activity against all immunoglobulin classes and light chains.:]: Granulocyte Antigens. Ten milliliters of whole blood were drawn into a heparinized tube containing 25 units of heparin/ml and gently mixed. One milliliter of Plasmagel§ was added and mixed. T h e specimen was then centrifuged for 8 minutes tMeloy #C-201, dilution 1:10. § Roger Bellon Laboratories, Neuilly, France.
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Routine Laboratory Procedures
A.J.CP.—Vol.
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LE-ANF INCONGRUITY
Varying Fixation. Various fixatives were used to fix leukocyte smears prior to serum application. These included ethyl alcohol (cold, 70%, for 10 minutes), ethyl alcoholether (50/50 dilution, cold, for 10 minutes), and acetone (cold and room temperature for 10 minutes). DNA Binding. DNA binding was done in the laboratory of Dr. Naomi Rothfield. 20 Results Both reviewers verified the laboratory's readings of all slides (26 of 26). T h e phagocytic cell was often likely to be a monocyte rather than a polymorphonuclear leukocyte. Table 1 summarizes the clinical diagnoses of the 20 patients. Six patients had rheumatoid arthritis, three had major d r u g reactions, two had tendonitis, and nine had other conditions. Table 2 summarizes the organ-system involvement and pertinent laboratory tests of the 20 patients and tabulates the provisional criteria 7 for systemic lupus erythematosus each fulfilled. Three patients (9, 14, and 17) each satisfied four or more criteria for systemic lupus erythematosus. Patient 9 had had dis-
coid lupus in the past, and had lymphopenia (leukocyte count 3,500), a butterfly rash, and arthralgias and pain on motion of both ankles. Patient 14 had degenerative joint disease of both knees, longstanding alopecia on one side of her head, and facial photosensitivity. Patient 17 had intermittent pleuritis, one episode of nasal ulceration, xerostomia, and Raynaud's phenomenon. Most of the other patients had multiple organ-system involvement, but their disease did not fulfill the rigid criteria needed for the diagnosis of systemic lupus erythematosus. These patients had involvement of the same organs commonly involved in systemic lupus erythematosus, but signs, symptoms, and laboratory values were not diagnostic of systemic lupus erythematosus and did not satisfy the American Rheumatism Association (ARA) criteria (Table 3). For example, renal involvement, to satisfy an ARA criterion, requires casts or proteinuria with values in excess of 3.5 g per day. Pyuria, hematuria, decreased creatinine clearance, or lesser proteinuria may occur in SLE, but do not satisfy the provisional ARA criteria. Specimens from 19 of 20 patients each had at least one tart cell or pseudo-LE cell. Eighteen of the 19 had pseudo-LE cells and tart cells equaling or outnumbering classic LE cells (Table 1 and Fig. 1). No patient of the nine with known SLE had a ratio of classic LE cells to pseudoLE cells of less than 1.0. Three patients (5, 6, and 18) had higher titers of ANF when the nuclear substrate was fixed prior to application of test serum (Table 1). One of these patients (18) had a positive ANF test result of 1:20 (unfixed) two months before her LE cell preparation was positive. However, by the time the LE cell preparation was performed, her ANF test result had become negative, and she had never received treatment. Seven patients (3, 4, 5, 6, 9, 12, 17) (35%)
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at 160 X g. T h e supernatant was removed and centrifuged at 475 X g for 6 minutes. The cells in the pellet were washed in PBS three times and centrifuged at 200 x g for 6 minutes after each wash. One drop of the final suspension was placed on a clean glass slide using a capillary tube and spread with a coverslip. The slides were air-dried and either used immediately or stored at 4 C for less than a week. Defibrinated leukocytes were prepared by vigorously shaking 10 ml of fresh whole blood for 10 minutes with glass beads and adding 1 ml Plasmagel, and treated as described above. Damaged leukocytes were created by allowing the final suspension of heparinized leukocytes to incubate at 4 C overnight. The cells were fixed (see below). Serum was then placed on the substrate and treated as in the ANF test described above.
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Table 1. Clinical Diagnoses and Experimental Results in Cases of 20 Patients with Positive LE Cell Preparations and Negative ANF Tests
Case
Diagnosis
Comments
Classic LE Pseudo LE and Tart Cells
Gain in Titer with Tissue Fixation
ANA in Low Titer
GS-ANF
Perivascular Fluorescence Titer 1:8
Probable rheumatoid arthritis
Seropositive
3/26
X
2
Probable rheumatoid arthritis
Seropositive
6/24
X
3
Definite rheumatoid arthritis
Seropositive
3/4
X
4
Definite rheumatoid arthritis
Seropositive
1/11
X
5
Definite rheumatoid arthritis
Seropositive
1/3
X
X
6
Definite rheumatoid arthritis
Seropositive
17/13
X
X
7
Drug reaction
Sulfamethoxazole
1/29
8
Drug reaction
9
Drug reaction
Chlorpropamide Sulfamethoxazole phenoxymethyl PCN
3/8 2/2
10
Tendonitis
6/11
11
Tendonitis
3/13
12
Avascular necrosis
13
Hypersensitivity pneumonitis Degenerative joint disease
14 15
Secondary to steroids ? Antigen Discharge diagnosis of SLE
X
X
X
X X
3/27
X
1/7
Discharge diagnosis of SLE
1/0
Discharge diagnosis of SLE
1/1
18
CVA with monoarticular arthritis
Discharge diagnosis of SLE
1/29
19
Skin rash
Discharge diagnosis of SLE
1/2
20
Magnesium deficiency
Alcoholic myopathy
3/27
17
X
1/6
Juvenile rheumatoid arthritis Recurrent juvenile rheumatoid arthritis in adult Sjogren's syndrome
16
X
X
1/29
X X
X
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1
Nonspecific abnormalities Deforming arthritis Dermatologic Renal Central nervous system Hematologic Per cent DNA binding
TOTAL CRITERIA
Provisional ARA criteria for SLE Arthritis without deformity Central nervous system Serositis Raynaud's phenomenon Mucous membrane ulcers Photosensitivity Facial erythema Discoid lupus Alopecia Hematologic Proteinuria Cellular casts False-positive serologic test for syphilis LE cells
X
12
X
12
X
X
8
X
X
3
X
X X 1 2
1 2
X 8
X
X 1 2
X
4
X
6
6
X X X X 10
X
X X 1 2
5
12
X X X
X 2
X
7
11
X
X 1
8
16
X X
X 5
X
X X
X
9
3
X
10
1
17
X
11
Patient
X 13
X
X 1 1
12
18
X 2
X
13
0
X
X
12
X
X
15
X 4
X
X X
14
X
33
X 30
X 4
X X X
17
X
X 1 2
X
16
9
X
X
X 2
X
18
8
X
X
19
20
6
X X
X 1 1
Table 2. Presence or Absence of Nonspecific Abnormalities and Signs, Symptoms, and Laboratory Values Composing the ARA Criteria for SLE in 20 Patients with Positive LE Cell Preparations and Negative ANF Tests
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to
o c •
z
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KOLLERET AL.
using leukocytes as a nuclear substrate. In both cases, these patterns were found with undiluted serum only. Varying the method of preparation and fixation of substrate did not affect the generally negative results for granulocyte-specific antinuclear factors (Fig. 2). Using defibrinated leukocytes did not increase sensitivity. Both patients were leukopenic. No patient had DNA binding within the range seen in active systemic lupus erythematosus. Discussion LE Preparation The positive LE cell test as currently defined necessitates the presence of at least two classic LE cells on the slide or one classic LE cell on each of two slides. Formation of the LE cell is a multi-stage process that requires four distinct elements: (1) nuclear substrate; (2) LE factor; (3) phagocytic cell; (4) complement. Most laboratories use a direct LE cell test using autologus
Table 3. Detailed Analysis of Nonspecific Organ-system Involvement in 20 Patients with Positive LE Cell Preparations and Negative ANF Tests System
Patient
Abnormality
CNS (3)
6 7 18
Mild depression Lethargy and coma Cerebrovascular accident age 34 years due to small-vessel occlusion
Hematologic (4)
4 6 12 16
Positive Coombs' test, not anemic, had previously received blood transfusion Unexplained anemia Unexplained anemia Unexplained anemia
Dermatologic (3)
7 8 9
Renal (10)
2 3 6 7 9 15 16 17 19 20
Total-body erythema Erythematous maculopapular and vesicular lesions on the trunk Longstanding frontal alopecia Trace proteinuria Trace proteinuria, pyuria Trace proteinuria, hematuria Pyuria, proteinuria, hematuria, decreased creatinine clearance Pyuria, proteinuria Pyuria Proteinuria (1 g), decreased creatinine clearance Pyuria, hematuria Proteinuria (430 mg), pyuria Pyuria
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had low titers of ANF that would ordinarily be considered negative by the laboratory. One patient (3) had a low titer of ANF at the time of the study, but within 6 months, the ANF test result became positive at a titer of 1:100. Specimens from nine patients had fluorescent patterns on the mouse liver that were not nuclear in position. T h e patterns were perivascular and sinusoidal (Table 1). T h e results of ANF testing were verified in the reference laboratory. 30 This laboratory routinely fixes mouse liver sections. T h e results were almost identical to the results obtained at the Medical College of Virginia. Three patients had titers of 1:16 or more. T h e same seven patients who had low titers in our laboratory and two additional patients had low titers of ANF using undiluted sera, and specimens from the same nine patients showed non-nuclear fluorescence. Specimens from two patients (7 and 13) had positive diffuse fluorescent patterns
A.J.C.P.—Vol.66
September 1976
501
LE-ANF INCONGRUITY ©
o=>>-#->!50
I 50
Clossic LE cells Pseudo LE cells plus tart cells
IOO
0 75 0 50 0 25
000
A •v Systemic Patients in Study Lupus (20) Erythematosus (9)
fl) only one abnormal cell seen on this slide
FIG. 1. Ratios of classic LE cells to pseudo-LE cells plus tart cells in LE preparations, 20 patients with the LE cell preparation-ANF test incongruity and nine patients with known SLE.
10
Substrate / Fixation / Dilution
15
20 _i
Liver / unfixed / 1: 2 0 Liver /acetone / 1:20
Fie. 2. Results of special ANF tests of 20 patients with the LE cell WBC /cold ETOH /(undiluted) preparation-ANF test incongruity. Conditions of the substrate, fixation technic, and dilution of serum are WBC /cold ETOH-ether /(undiluted) shown on the left. The number of patients tested with each technic and the number of positive tests Aged WBC /cold ETOH /(undiluted) (shaded area) are denoted by bars. WBC/acetone /(undiluted)
positive negative
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cells as the source of nuclear material. However, when the patient is leukopenic, homologous or even heterologous cells may be used in an indirect test. The nuclear substrate from certain animal sources may, in fact, be more sensitive to the action of LE factor than human substrate. 5 It has also been shown that either granulocytes 19 or lymphocytes 25 lend themselves to use as nuclear substrate in the LE cell test. More important than the source of the nuclear material is its viability. The LE factor will react only with nuclei 23 and, therefore, the factor must penetrate the cytoplasmic membrane. Diffusion of the LE factor across the cytoplasmic membrane can be facilitated by damaging that membrane. This can be effected by: vigorous shaking with glass beads; crushing and straining a clot through a fine-mesh sieve; drying the cells; freezing and thawing the cells; or chemical injury. The factor does not affect living cells,27 and hence it is rare to find an LE cell in vivo except in those special cases where numerous damaged cells are in a closed space and cannot
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ROLLER ET AL.
Other variables also affect the LE cell preparation. High concentrations of heparin will inhibit the LE phenomenon. Additionally, an adequate incubation time is required. The mechanism of the LE phenomenon was studied in detail by Robineaux. 29 On adding the lupus factor to substrate nuclei, changes are evident almost instantly. As the
nuclei are homogenizing, the cytoplasmic granules become perinuclear. Finally, the cytoplasmic membrane ruptures, and only then do other polymorphonuclear cells become attracted to the affected nucleus. T h e nucleus must be in contact with the environment for this process to occur. T h e phagocytic cell will engulf the free or exposed nucleus, leaving the cytoplasm unincorporated. Tart cells and pseudo-LE cells may resemble LE cells. Tart cells, described at the same time the LE cell was described, 16 were named after the patient in whom they were found. T h e mechanism of their formation is not as well worked out as that of the LE cell. Kurnick 18 has observed that pseudo-LE cells can be formed as a result of an entire cell's being phagocytized. As intracytoplasmic breakdown occurs, the nucleus of the inclusion cell becomes more homogeneous. The reticular pattern is lost and a basophilic ring appears around the border of the inclusion. Rarely, all staining differences between the LE nucleus and the pseudo-LE nucleus may be lost, either by the above-described mechanism or by another, unknown, mechanism, and the cell may mimic a classic LE cell.15 Tart and pseudo-LE cells have been produced in the presence of leukoagglutinins. 12 Indirectly supporting this concept is the fact that pseudo-LE cells are found in patients with drug reactions, 22 which may also be associated with antibodies to blood components. Forty per cent of patients who have SLE may have luekoagglutinins present. 8,10 T h e problem with interpreting data regarding leukoagglutinins lies in the lack of standardized procedures and the lack of differentiation between autoantibodies and isoantibodies. It is also difficult to distinguish between antibodies directed against surface antigens and immune complexes nonspecifically bound to the cell membrane. T h e presence of factors directed against lymphocytes has been well-established. 24
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be easily cleared. Such spaces include joint, 17 pericardial, 31 and pleural 32 cavities. T h e LE factor is usually a 7S IgG. Other anti-deoxynucleoprotein antibodies that are not IgG but instead IgM have been found. These, however, have less tendency to form LE cells because of their low affinity for complement. 4 When the LE factor combines with the nuclear substrate, the chromatin pattern of the nucleus is gradually lost, and eventually the nucleus becomes homogeneous. The homogeneous mass is then exposed to the environment, either partially through a slight disruption of the cytoplasmic membrane, or totally by extrusion of the mass. Once feee, it is called a "hematoxylin body." In order for the hematoxylin body to be engulfed by another phagocyte a serum factor must be present. This factor is thought to be complement because it can be removed by heating the LE serum to 56 C for 30 minutes and because it can be deactivated by anti-C3. Exacdy how much complement is necessary for the completion of the LE test is unknown, but it is thought to be very little.13 Without complement, many hematoxylin bodies are found. Hematoxylin bodies by themselves are not diagnostic, but they are often seen in conjunction with LE cells. On the other hand, in the presence of complement, the hematoxylin body can be phagocytized by polymorphonuclear leukocytes (occasionally either a less mature cell of the myelocytic series or a monocyte) to form an LE cell. T h e phagocytizing cell will become swollen and its nucleus will become displaced into a peripheral position.
A.J.C.P. —Vol. 66
September 1976
LE-ANF INCONGRUITY
Antinuclear Factor Having established the classic LE cells were present, and setting aside the concept of a false-positive LE cell preparation for the moment, the issue of a falsenegative ANF test result was raised. It seemed appropriate to review the mechanisms of the ANF test and the possible ways by which false-negative results can occur. T h e positive ANF test is a threelayered test: Layer 1, nuclear substrate;
layer 2, patient's serum containing antinuclear factors; layer 3, fluorescein-conjugated anti-human immunoglobulin, monovalent or polyvalent. Tissues that have been utilized to supply nuclear material include kidney (mouse and rat), liver (mouse and rat), tumor imprints, buccal mucosa, spleen imprints, tissue culture cells, chick erythrocytes, human leukocytes, and calf thymus. Once a tissue section is placed on the slide, the section may or may not be fixed. Several mild fixatives are in use, acetone and ethyl alcohol being the most common. It had been shown by Beck2 and later by Claymaet and Nakamura 6 that without fixation, some soluble nuclear antigens would be lost. We had three patients whose titers of ANF increased as a result of fixing. Two had finely speckled patterns and one had a diffuse pattern. There is no standard method for ANF determinations. One is clearly needed, and we feel that fixation should be a part of any standard procedure. Layer 2 is the patient's antinuclear factors). There are three ways in which the patient may have antinuclear factor but a negative antinuclear factor test result: (1) ANF present but entirely bound to circulating antigen; (2) ANF present and unbound, but titer too low to be discovered at screening dilution; (3) ANF present but organspecific, i.e., it can react with nuclei of leukocytes, giving a positive LE cell preparation, and not react with other tissue antigens, giving a negative ANF test result. It has been shown that immune complexes, when present, may produce falsenegative ANF test results. 3 In such a case, when the serum undergoes acid dissociation of the immune complexes, the ANF test result becomes positive. It is unlikely that such a mechanism was operative in our patients. The LE cell preparations were positive, implying the presence of circulating unbound antinuclear factors. In addition, one would expect that the presence of the immune complexes would
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Whether these factors are a primary or secondary phenomenon is still under investigation. The theory presented regarding the formation of the pseudo-LE cells has been thought by some to be oversimplified. 21 Several investigators 21 have reported that tart and pseudo-LE cells may precede the development of the classic LE phenomenon. We have seen free cell nuclei that resemble the inclusion of the pseudo-LE cell in LE cell preparation slides. It must be remembered that in evaluating studies where the LE cell is of critical importance, stringent criteria for the identification of the LE cell must be strictly observed. There have been several reports in the literature where the LE cell appeared to have been a pseudo-LE cell. After careful review of the 20 cases reported in this paper, the predominant cell was found to be the pseudo-LE cell or tart cell in specimens from 18 of the 20 patients, whereas the opposite was the case in all nine SLE specimens. We believe that LE cell preparation in which pseudo-LE cells and tart cells greatly outnumber classic LE cells cannot be used to verify the diagnosis of SLE. Consequently, we recommend that the characteristics of the entire slide should be examined. One or two classic LE cells do not, by themselves, in a milieu of tart and pseudo-LE cells, have diagnostic significance, because they may have attained their morphology by the leukoagglutination pathway.
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Finally, the fluorescent conjugate may be responsible for a falsely negative result. If, for example, the ANF is an IgM, as it may be in 4% of patients, 14 and the conjugate is monovalent for IgG, no fluorescence will be evident. Special polyvalent conjugate did not increase the yield of positive ANF tests. An interesting finding was the identification of nine patients whose specimens showed a perivascular sinusoidal pattern at a dilution of 1:8 or more. This pattern is similar to that seen with antivascular antibody 26,34 and antireticulin antibody. 28 T h e diagnoses of our 20 patients varied. Rheumatoid arthritis seemed to predomi-
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nate, but this could be due to the fact that the sera had been obtained from the rheumatoid arthritis serology laboratory. No clear clinical pattern was evident. All the patients, however, seemed to have signs, symptoms, and laboratory findings that suggested systemic disease. The findings were similar, but not identical, to those that the ARA enumerates for systemic lupus erythematosus (Table 2). Due to inappropriate application of the ARA criteria, five patients initially had been incorrectly diagnosed as having SLE. Summary The cases of 20 patients who had positive LE cell preparations and negative ANF test results were studied. The reasons for this serologic paradox are discussed. Three patients had false-negative ANF test results because the sections were not fixed and the soluble antigens were lost during the performance of the test. One patient had a titer of ANF that was below the screening level but was subsequently elevated several months later. The problem of a falsepositive LE cell preparation is discussed. Other mechanisms are discussed and rejected as reasons for the serologic abnormalities. No clearly identifiable syndrome emerges from clinical analysis of these 20 cases. The following conclusions can be drawn, based on our experience: (1) T h e nuclear substrate in the ANF test should be fixed. (2) Although we do not recommend full titering of the ANF for every patient, there will be an occasional case in which it would be helpful. (3) When classic LE cells are greatly outnumbered by pseudo-LE cells or tart cells, or both, a positive LE cell preparation has limited diagnostic value, and should not be used to verify a diagnosis of SLE or to fulfill the provisional ARA criteria. (4) With the limitations and mechanisms of the ANF test and LE cell preparation understood, it is justifiable to use the ANF test as a screening test for connective-tissue diseases. However, when because of technical considerations the ANF test is not as reliable as it might be, the LE cell prepara-
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bind complement and hence lower its serum concentration. T h e C3's of the patients for whom this information was available were normal. In addition, DNA binding was in the normal range for all patients (Table 2). One patient had a titer of ANF below the dilution screening point and later developed a more than fourfold increase in titer. When the clinical or laboratory picture warrants it, we believe the ANF test should be done at lower dilutions. T h e existence of granulocyte-specific ANF has been known since 1964.11 These factors react preferentially with the nuclei of granulocytes. They are measured using leukocytes as a nuclear source, by finding a higher titer present against these leukocytes than against human thyroid cell nuclei. Granulocyte-specific ANF have been found in association with rheumatoid arthritis, especially with leukopenia and/or Felty's syndrome. In general, we have found that human leukocytes in smears are less sensitive than mouse liver as a nuclear antigen source. We found two patients who had low titers of granulocytespecific antinuclear factors, but positive tests were obtained using different fixative technics and were of low titer, the results being weakly positive in undiluted sera. These two patients both had leukopenia. Varying the fixative and preparation technic did not increase the yield of granulocyte-specific ANF.
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A morphological study. South Med J 49:560566, 1956 16. Hargraves MM, Richmond H, Morton R: Presentation of two bone marrow elements: The "tart" cell and "L.E." cell. Proc Staff Mayo Clin 23:25-28, 1948 17. Hunder GG, Pierre RV: In vivo LE cell formaAcknoxukdgments. Mrs. Nellie Curry, Mrs. Loretta tion in synovial fluid. Arthritis Rheum 13:448Albert, and Mrs. Carolyn Jones provided technical 450, 1970 assistance. Dr. Shaun Ruddy and Dr. Keith Whaley offered suggestions and encouragement. Help and 18. Kurnick NB: The pseudo-LE cell phenomenon with report of a case. Blood 12:382-390, guidance were provided by Dr. Naomi Rothfield of 1957 the University of Connecticut School of Medicine. 19. Larson RA, Boyum A: Development of an indirect LE factor test. Acta Med Scand 543: References 21S-29S, 1972 1. Barnett EV, Rothfield N: The present status of 20. Luciano A, Rothfield NF: Pattern of nuclear antinuclear antibody serology. Arthritis Rheum fluorescence and DNA-binding activity. 12:543-544, 1969 Ann Rheum Dis 32:337-341, 1973 2. Beck JS: Variations in the morphological patterns 21. Marmont A: Nucleolytic phagocytosis (L.E. cell of "autoimmune" nuclearfluorescence.Lancet phenomenon) in systemic lupus erythematosus, 1:1203-1205, 1961 rheumatoid arthritis, and systemic sclerosis, 3. Blomjous FJEM, Feltkamp-Vroom TM: Hidden First International Symposium on Immunoantinuclear antibodies in seronegative systemic pathology. Edited by Grabar P, Miescher P. lupus erythematosus patients an in NZB and Basel, Benno Schwabe and Co. Verlag, 1958, (NZB X NZW) Fl mice. Eur J Immunol pp 479-502 1:396-398, 1971 22. Meyler L: Phagocytosis in drug allergy. Acta 4. Blondin C, McDuffie FC: Role of IgG and IgM Med Scand 150:33-38, 1954 antinuclear antibodies in formation of lupus 23. Mieschner P, Fauconnet M: L'absorption du erythematosus cells and extracellular material. facteur "L.E." par des noyaux cellulaires Arthritis Rheum 13:786-797, 1970 isoles. Experientia 10:252-254, 1954 5. Carrera AE, Reid MV, Kurnick NB: Differences 24. Mittal KK, Rossen RD, Sharp JT, et al: Lymphoin susceptibility of polymorphonuclear leukocyte cytotoxic antibodies in systemic lupus cytes from several species to alteration by erythematosus Nature (Lond) 225:1255systemic lupus serum: Application to a more 1256, 1970 sensitive L.E. phenomenon. Blood 9:1165- 25. Moyer JB, Fisher GS: Experimental production 1171, 1954 of "L.E." cells. Am J Clin Pathol 20:1011-1015, 6. Cleymaet JE, Nakmura RM: Indirect immuno1950 fluorescent antinuclear antibody tests: Com- 26. Person DA, Leatherwood CM, Sharp JT: Antiparison of sensitivity and specificity of different vascular antibody. Ann Rheum Dis 33:371substrates. Am J Clin Pathol 58:388-393, 375, 1974 1972 27. Rapp F: Localization of antinuclear factors from 7. Cohen AS, Reynolds WE, Franklin EC, et al: lupus erythematosus sera in tissue culture. Preliminary criteria for the classification of J Immunol 88:732-740, 1962 systemic lupus erythematosus. Bull Rheum Dis 28. Rizzetto M, Doniach D: Types of "reticulin" 21:643-648, 1971 antibodies detected in human sera by immuno8. Dausset J, Colombani J, Colombani M: Study of fluorescence. J Clin Pathol 26:841-851, 1973 leukopenias and thrombocytopenias by the 29. Robineaux R: Study of LE cell formation by direct antiglobulin consumption test on leukophase contrast microcinematography, Mechcytes and/or platelets. Blood 18:672-690, 1961 anisms of Hypersensitivity. Edited by Saffer 9. Dubois EL: Lupus Erythematosus. Los. Angeles, JH, LoGrippo GA, Chase MW. Boston, California, University of Southern California Little, Brown and Company, 1959, pp 371Press, 1974, p 487 395 Engelfriet CP, Van Loghem JJ: Studies on leuco10 NF, Fragione B, Franklin EC: Slowly cytes iso- and auto-antibodies. Br J Haematol 30. Rothfield sedimenting mercaptoethanol-resistant anti7:223-238, 1961 nuclear factors related antigenically to "M" 11 Faber V, Elling P, Norup G, et al: An antiimmunoglobulins (ym-globulin) in patients nuclear factor specific for leucocytes. Lancet 2: with systemic lupus erythematosus. J Clin 344-345, 1964 Invest 44:62-72, 1965 12 Finch SC, Ross JF, Ebaugh FG: Immunologic mechanism of leukocyte abnormalities. J Lab 31. Seaman AJ, Christeron JW: Demonstration of L.E. cells in pericardial fluid. JAMA 149:145Clin Med 42:555-569, 1953 147, 1952 Golden HE, McDuffie FC: Role of lupus erythe13 matosus factor and accessory serum factors in 32. Ibid: See footnote Grondahl R production of extracellular nuclear material. 33. Souhami RL, Homma M, Gumpel JM, et al: The absence of antinuclear factors (by immunoAnn Intern Med 67:780-790, 1967 fluorescence) in certain patients with abnormal 14 Gonzalez EN, Rothfield NF: Immunoglobulin L.E. cell tests. Arthritis Rheum 8:470, 1965 class and pattern of nuclear fluorescence in systemic lupus erythematosus. N Engl J Med 34. Tan EM, Pearson CM: Rheumatic disease sera 274:1333-1338. 1966 reactive with capillaries in the mouse kidney. 15. Gouldin EN, Diggs LW: L.E. and L.E.-like cells: Arthritis Rheum 15:23-28, 1972 tion should be performed in addition. (5) T h e ARA criteria, when used to support the diagnosis of systemic lupus erythematosus, should be employed with precision.
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