P ERSPECTIVES IN LUPUS NEPHRITIS J NEPHROL 2002; 15 (suppl.

6): S20-S27

The use of laboratory tests in diagnosis and

monitoring of systemic lupus erythematosus
Renato Alberto Sinico, Bruna Bollini, Ettore Sabadini, Luca Di Toma, Antonella Radice

Nephrology and Dialysis Unit and Center of Clinical Immunology and Rheumatology, San Carlo Borromeo Hospital,
Milan - Italy

ABSTRACT: Clinical immunology laboratories play an essential role in diagnosis and monitoring of systemic lupus ery-
thematosus (SLE). To obtain the best results in terms of diagnostic performance and clinical usefulness, the following
recommendations should be fulfilled:
- Indirect immunofluorescence on Hep-2 cells remains the method of choice for the detection of anti-nuclear antibod-
ies (ANA). The sensitivity of ANA test for SLE is very high (almost 100%) but its specificity low since ANA can be pre-
sent in a number of different clinical conditions and even in normal controls.
- Anti-dsDNA antibodies are highly specific for SLE and present in a high proportion of SLE patients (40-80%). The
method of choice for anti-ds DNA is the Farr assay; however, the necessity of using radioactive material decreases its
applicability. As an alternative, immunofluorescence on Crithidia Luciliae can be used in the diagnostic phase for its
high specificity. It is not advisable to use ELISA, in the diagnostic phase, due to its low specificity. The quantitative de-
termination of anti-dsDNA is useful for monitoring patients, in particular in the presence of nephritis. For monitoring,
a quantitative method should be used (Farr assay or ELISA).
- The detection of antibodies to extractable nuclear antigens (ENA) and to phospholipids (Lupus anticoagulant and an-
ti-cardiolipin antibodies with a β2 glycoprotein I-dependent method) are useful to identify subgroups of patients at risk
for some clinical manifestations (i.e. anti-phospholipid syndrome).
- New assays (anti-C1q and anti-nucleosome antibodies) have been recently proposed for diagnosis (anti-nucleosome)
and monitoring SLE patients (anti-C1q and anti-nucleosome antibodies), with promising results.
- Among biological parameters, urinary levels of monocyte chemoattranct protein 1 (MCP1) seem to be the most use-
ful to monitor nephritis activity in lupus patients.

Key words: Systemic lupus erythematosus (SLE), Anti-nuclear antibodies (ANA), Anti-dsDNA antibodies, Anti-extractable nuclear
antigens (ENA) antibodies, Anti-phospholipid antibodies, Anti-nucleosome antibodies, Anti-C1q antibodies

INTRODUCTION Indeed, the role of the clinical immunology laborato-

Laboratory, and particularly clinical immunology lab-
oratories, play an essential role in diagnosis and mon-
itoring of systemic lupus er ythematosus (SLE) as
demonstrated by:
- Updated American College of Rheumatology (ACR)
criteria for the diagnosis of SLE which include sev-
eral autoantibodies (Tab. I).
- Anti-DNA antibodies and serum complement levels
used to evaluate disease activity (Tab. II).
- Some autoantibodies, i.e. antiphospholipid antibod-
ies, associated with peculiar clinical manifestations
(Tab. III).
ry in SLE is:
1. to confirm or exclude diagnosis
2. to monitor disease activity
3. to identify subgroups of patients.
With such an important key role clinical immunology
laboratories should be able to provide assays charac-
terized by:
- High sensitivity (close to 100%)
- High specificity (close to 100%)
- High positive and negative predictive values.
Currently no one test can satisfy all these require-
ments because increasing in specificity usually leads to
reduced sensitivity.

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Sinico et al

Other factors which contribute to unsatisfactory re- All these factors can greatly influence the interpreta-
sults are: tion of the results and their clinical meaning.
- Different assays are used for the same antibody (im- Different assays detect peculiar antibody properties
munofluorescence, ELISA, radioimmunoassay, im- and it is quite possible that the same autoantibody
munoblotting). reacts differently according to method used.
- Different antigenic substrates (nuclear extracts, pu- Therefore, it is important for the clinician to be aware
rified antigens, recombinant proteins) can be used of the main features of the assay in order to correctly
for the same method (i.e. ELISA). interpret the results.
- Isotypes of detected antibody can be different: some The assays traditionally used in the diagnosis and
kits utilize anti-IgG antiserum, others anti-im- monitoring of SLE are:
munoglobulin antisera, which detect all Ig isotypes. - Anti-nuclear antibodies (ANA)
- Antibodies with different affinity/avidity react dif- - Anti-double stranded DNA (anti-dsDNA)
ferently in different assays. - Anti-histone antibodies
- Methods are often not standardized; reference sera - Anti-extractable nuclear antigens antibodies (ENA)
and quality control programs are not always available. - Anti-ribosomal P protein
- Normal range values can be determined by various - Anti-phospholipid antibodies (aPL).
methods: mean ± 2 or 3 standard deviations, per-
centiles, receiver operating characteristic (ROC) curve.
ANTI-NUCLEAR ANTIBODIES (ANA)
TABLE I - ACR CLASSIFICATION CRITERIA FOR SLE (UP-
The methods currently used for ANA detection are:
DATED 1997)
indirect immunofluorescence (IIF) and, more recent-
1) Malar rash ly, ELISA. In the past, the most common substrates for
2) Discoid rash immunofluorescence were rodent tissues (i.e. liver
3) Photosensitivity and kidney). Subsequently, they have been substituted
4) Oral ulcers with a human epithelial cell line (Hep-2).
5) Arthritis Hep-2 cells produce more positive ANAs than rodent
6) Serositis tissues, and some ANAs (for example, anticentromere
7) Renal disease antibodies) can only be reliably detected on Hep-2
8) Neurological disorders substrate.
9) Hematological disorders ANA screening tests have high sensitivity (more than
95%) but low specificity and predictive value (10-
10) Immunological abnormalities 40%). Thus, they are used mainly in the exclusion of
a. anti-dsDNA Ab
SLE and to assess the need for further testing for spe-
b. anti-Sm Ab
c. lupus anticoagulant and/or anti-cardiolipin Ab and/or
cific antibodies (anti-dsDNA and anti-ENA). In-
false-positive serologic test for syphilis creased sensitivity results from the expression of more
relevant nuclear antigens in the human cells.
11) Positive antinuclear Ab (ANA) ELISAs have recently been introduced for ANA detec-
tion. The kits, commercially available, utilize different

TABLE II - SLE DISEASE ACTIVITY INDEX (SLEDAI)

• Seizure • Proteinuria
• Psychosis • Pyuria
• New malar rash

• Organic brain syndrome • Alopecia

• Visual disorders • Mucous membrane ulcers

• Cranial nerve involvement

• Lupus headache • Pleurisy
• Cerebrovascular accident • Pericarditis
• Vasculitis • Low complement
• Arthritis • Increased DNA binding
• Myositis • Fever
• Casts • Thrombocytopenia
• Hematuria • Leukopenia

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Laboratory tests in diagnosis and monitoring of SLE
TABLE III - POSSIBLE ASSOCIATION OF AUTOANTIBODIES WITH CLINICAL FEATURES
AutoAb target Frequency (%)
dsDNA 30 – 70
Ro 10 – 15
Ribosomal P 5 – 10
Histone 50 – 80
ACA 30 - 50
TABLE IV - CORRELATION AMONG THREE ANTI-DNA ASSAYS
Anti-DNA assay Crithidia Luciliae ELISA
Farr assay r = 0.85; r = 0.67;
p < 0.001 p < 0.001
Crithidia Luciliae r = 0.70;
p < 0.001
modified from Smeenk RJK et al, 1996.
antigenic substrates, from nuclear extracts to mixtures
of purified and/or recombinant antigens.
ELISAs are easy to perform, are semi quantitative, can
be automized and are cost-effective.
Nevertheless, at present, use of ELISA for the ANA test
has not been subject to widespread population testing
and has some disadvantages in comparison with im-
munofluorescence:
- The nuclear antigens used, both from purified
and/or recombinant origin, do not always have the
normal tertiary structure that intact cells do.
- Not all the antigen specificities have been identified.
Moreover, the variability of the antigenic preparations,
used in the different kits, gives problems of repro-
ducibility (1). In a recent paper (2), where seven differ-
ent commercially available kits were compared, the
sensitivity varied from less than 70% to more than 90%.
In conclusion, immunofluorescence on Hep-2 cells
remains the method of choice for ANA detection. Sen-
sitivity is very high (close to 100%) but specificity and
positive predictive value are low, since ANA may be
present in different diseases and even in normal sub-
jects. A negative ANA test makes the diagnosis of SLE
very unlikely whereas a positive ANA indicates further
testing is necessary.
Specificity can be improved using an anti-IgG instead
of an anti-immunoglobulin antiserum and taking into
account the ANA titre: whereas low titre may be found
in a large number of normal subjects, especially with
increasing age, titre higher than 1/160 are found al-
most exclusively in connective tissue diseases. ANA
titre, although important for diagnosis, is not useful
for monitoring (3-4).
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Clinical association
Nephritis
Sjögren’s / skin / neonatal lupus / cong. heart block
Neuropsychiatric SLE
drug induced SLE
APL syndrome
ANTI-DSDNA ANTIBODIES
Anti-dsDNA antibodies are highly specific for SLE
and are associated with renal involvement; anti-sin-
gle stranded (ss) DNA antibodies on the other hand
may be found in several different clinical conditions
(5-7).
There are several problems in the detection of anti-
dsDNA, including:
- Substrate differences: i.e. DNA from calf thymus,
DNA from plasmid or from other sources (8).
- The isotype of antibody detected: Farr assay can de-
tect all Ig classes whereas ELISAs usually detect only
IgG antibodies.
- Antibody affinity: Farr assay detects high affinity
antibodies whereas ELISAs both high, medium and
low affinity antibodies.
- Problems with standardization.
- Assay specific parameters.
Three different methods are currently available for
anti-dsDNA antibodies detection: indirect immunoflu-
orescence on chritidia luciliae (CLIF), Farr assay,
which is a radioimmunoprecipitation test, and ELISA.
1. CLIF has a high specificity but medium-low sensitiv-
ity and detects antibodies of intermediate to high
avidity.
2. Farr assay has high specificity and good sensitivity
for SLE. It measures mainly antibodies of high avid-
ity which are pathogenic (9).
3. ELISAs have a very high sensitivity but low specifici-
ty for SLE and can detect antibodies of low to high
avidity. ELISA specificity is largely influenced by the
antigenic substrate used. Therefore, the results ob-
tained with different ELISA kits are hardly compa-
rable (10).
As already mentioned, it is important for the clinician
to be aware of the method used in order to evaluate
the clinical significance of the result (11-17) (Tab. IV).
To summarize:
- As an aid to the diagnosis of SLE a very specific assay
should be preferred (Farr assay or alternatively
CLIF).
- For monitoring activity a quantitative assay should
be used (Farr or ELISA assay) (18, 19).
Sinico et al
ANTI-HISTONE ANTIBODIES
About 60-70% or more of SLE patients have anti-hi-
stone antibodies. However, these antibodies may be
found in a variety of other diseases. Thus, anti-his-
tone antibodies are not important for the diagnosis
of SLE, with the exception of drug induced SLE
(20, 21).
Titres of anti-histone antibodies may reflect disease ac-
tivity but it is not clear if they add much to other more
commonly performed assays, such as anti-dsDNA or
complement levels.
ANTIBODIES TO ENAS
Autoantibodies to ENAs are directed against several
nuclear antigens, among which the ones of clinical rel-
evance are:
anti-Sm
anti-RNP
anti-SSA/Ro
anti-SSB/La
anti-Scl70
anti-Jo1
Anti-Sm antibodies are highly specific for SLE and are
therefore included into ACR criteria.
They are found, however, in a minority of patients (15-
30%) and their presence is not related to particular
clinical manifestations.
Anti-RNP antibodies are not specific for SLE and can
be found in other connective tissue diseases. Their
presence at high titre is one of the criteria for diagno-
sis of the so-called mixed connective tissue disease.
Their presence in SLE patients is not related to specif-
ic signs or symptoms, with the exception of Raynaud
phenomenon.
Antibodies to SSA and to SSB are also found in pa-
tients with primary Sjögren’s syndrome.
They are found in about 25-50% of SLE patients.
SLE patients with anti-SSA and/or anti-SSB antibod-
ies develop, more often than SLE patients without,
“sicca syndrome”, subacute cutaneous lupus, neona-
tal lupus and congenital heart block.
Antibodies to Scl70 and antibodies to Jo1 are not
usually found in SLE patients but in scleroderma and
dermato-polymyositis respectively.
Several different methods are available for anti-ENAs
detection. Among them, ELISA, immunoblotting,
double immunodiffusion and immunoblotting.
None of them is ideal and sometimes it is necessary
to utilize more than one technique.
Titres of anti-ENAs antibodies can fluctuate with dis-
ease activity and treatment, but serial monitoring
does not effectively predict relapse.
RIBOSOMAL P ANTIBODIES
Anti-ribosomal antibodies are associated with neu-
ropsychiatric manifestations in SLE patients. They
are found in around 15% of patients. However, their
predictive value remains controversial.
ANTI-PHOSPHOLIPID ANTIBODIES (APL)
Anti-phospholipid antibodies are a heterogeneous
group of autoantibodies that exhibit a broad range
of target specificities, all recognizing various combi-
nation of phospholipids, phospholipid-binding pro-
teins or both.
Antiphospholipid antibodies interfere with coagula-
tion mechanisms resulting in a pro-thrombotic state.
The most commonly detected subgroups of aPL are
Lupus Anticoagulant (LAC), anti-cardiolipin anti-
bodies and anti-β2 glycoprotein I antibodies.
Antiphospholipid antibodies have been recently in-
cluded into ACR criteria for SLE (22).
The true target of aPL is represented by the complex
phospholipid-β2 glycoprotein I or even by the sole β2
glycoprotein I.
Indeed, anti-β2 glycoprotein I antibodies seem to cor-
relate better than anti-cardiolipin antibodies with the
clinical manifestations of anti-phospholipid syndrome
(vascular thrombosis and complications of pregnancy).
LAC and/or antiphospholipid antibodies are found
in 30-40% of lupus patients.
Anti-phospholipid antibodies are also found in pa-
tients with the “so-called” primary anti-phospholipid
syndrome.
There is a not complete overlap between LAC, mea-
sured by a functional assay, and anti-phospholipid
antibodies, measured by ELISA. Therefore, both as-
says should be performed.
The presence of LAC and/or aPL antibodies, espe-
cially if at medium-high titre, is associated with the
specific clinical manifestation of anti-phospholipid
syndrome (arterial/venous thrombosis and miscar-
riages) (23).
It should be pointed out however, that not all the pa-
tients with aPL antibodies develop the typical throm-
botic and pregnancy-associated manifestations.
On the other hand, thrombosis can occur in SLE in
the absence of aPL antibodies.
Higher titres of aPL antibodies are generally found
in the phases of disease activity. However, serum titre
of aPL antibodies is not particularly useful for moni-
toring disease activity.
Recently, new autoantibodies, which are associated
with thrombotic manifestations, have been described,
i.e. anti-phrotrombin antibodies. Their precise clini-
cal significance is still the object of intensive research.
S23
Laboratory tests in diagnosis and monitoring of SLE

Fig.1 – Algorithm for the diagnosis of SLE. Fig. 2 – Algorithm for SLE monitoring.

“NEW” AUTOANTIBODIES TABLE V - CORRELATION BETWEEN RENAL FLARES AND

IMMUNOLOGICAL DATA
Recently, several new autoantibodies have been re-
ported in SLE patients. Among them, the most inter- r p
esting ones are:
- anti-nucleosome antibodies
Ab anti-C1q 0.54 0.0001
- anti-C1q antibodies
- anti-endothelial cell antibodies Ab anti-dsDNA 0.38 0.0003
- anti-neutrophil cytoplasmic antibodies (ANCA)
- anti-SR antibodies C3 0.36 0.0004
- anti-telomere antibodies. C4 0.30 0.003

ANTI-NUCLEOSOME ANTIBODIES

Anti-nucleosome antibodies, which are responsible ANTI-C1Q ANTIBODIES

for the “so-called” LE phenomenon, play an im-
portant role in the pathogenesis of SLE (24-26).
The nucleosomes are the fundamental unit of
chromatin and consist of a core particle composed
of an octamer of 2 copies each of histones H2A,
H2B, H3, H4, around which is wrapped a stretch of
helical DNA.
Recently, a growing interest on anti-nucleosome
antibodies as a diagnostic tool has emerged in the
literature (27-29). Anti-nucleosome antibodies oc-
cur in 56-72% of SLE patients (27-29).
Conflicting results have however been reported
concerning the presence of anti-nucleosome anti-
bodies in other connective tissue diseases (27-29)
and their correlation, in lupus patients, with dis-
ease activity and renal involvement (28, 29). These
different results might be due to the different anti-
genic preparations used (28, 29).
The presence in lupus sera of a low molecular
weight component, able to bind to C1q, was de-
scribed more than 30 years ago by Agnello et al
(30).
However, only in more recent years, this compo-
nent has been identified as an IgG autoantibody
(31). In addition to SLE, anti-C1q autoantibodies
may occur in other diseases, such as rheumatoid
arthritis and membranoproliferative glomeru-
lonephritis.
Their presence and levels are related, in SLE, to re-
nal involvement and nephritic flares, even more
than in other laboratory assays such as anti-dsDNA
antibodies (32-37).
Therefore laborator y assays that reveal anti-C1q
antibodies seem to predict more accurately renal
involvement and flares (Tab. V).

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Sinico et al
OTHER AUTOANTIBODIES
Anti-endothelial cell antibodies, occurring in a
high percentage of SLE patients, and ANCA, pre-
sent in a minority of them, do not seem to have a
specific clinical significance (38-39).
Only very recently, anti-SR (phosphorilated pro-
teins), anti-guanosine and anti-telomere antibodies
have been described in lupus patients (40). Their
clinical significance remains to be elucidated.
BIOLOGICAL PARAMETERS
In addition to the described autoantibodies, also a
number of biological parameters have been used to
monitor disease and/or nephritis activity. Among
them, complement degradation products (urinary
C3d) (41) and endothelial cell damage markers, such
as thrombomodulin (42).
Several plasma and urinar y cytochines and
chemokines have been proposed as an aid to monitor
clinical activity (43-45). The most promising one
seems to be MCP-1 (monocyte chemoattractant pro-
tein 1). Its urinary levels correlate, in fact, strictly to
nephritis activity (44, 45).
CONCLUSIONS
To conclude, it is possible to give some clear indica-
tions to better utilize the clinical immunology labora-
tory in the management of SLE patients.
- Indirect immunofluorescence on Hep-2 cells re-
mains the method of choice for ANA detection. In
the case of ANA negativity, the diagnosis of SLE is
very unlikely.
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Address for correspondence:
R.A. Sinico, M.D.
Divisione di Nefrologia
Ospedale S. Carlo Borromeo
Via Pio II, 3
20153 Milano, Italy
renato.sinico@oscb.sined.net
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