ARTHRITIS & RHEUMATISM
Vol. 50, No. 11, November 2004, pp 3596–3604
Measurement of Erythrocyte C4d and Complement Receptor 1
in Systemic Lupus Erythematosus
Susan Manzi,1 Jeannine S. Navratil,2 Margie J. Ruffing,2 Chau-Ching Liu,2
Natalya Danchenko,1 Sarah E. Nilson,2 Shanthi Krishnaswami,3 Dale E. S. King,2
Amy H. Kao,2 and Joseph M. Ahearn2
Objective. C4-derived activation fragments are
the only complement ligands present on the surfaces of
normal erythrocytes. The significance of this observa-
tion is unknown, and the role of erythrocyte-bound C4
(E-C4) in human disease has not been explored. More
than any other human disease, the pathogenesis of
systemic lupus erythematosus (SLE) has been charac-
terized by defects in clearance of complement-bearing
immune complexes via erythrocytes expressing comple-
ment receptor 1 (CR1). This study was undertaken to
determine whether these functional defects might be
reflected by abnormal patterns of E-C4 and E-CR1
expression on erythrocytes of patients with SLE.
Methods. We conducted a cross-sectional study of
100 patients with SLE, 133 patients with other diseases,
and 84 healthy controls. Erythrocytes were character-
ized by indirect immunofluorescence and by flow cytom-
etry for determination of levels of C4d and CR1.
Results. Patients with SLE had higher levels of
E-C4d and lower levels of E-CR1 than did patients with
other diseases (P < 0.001) or healthy controls (P <
Supported by grants from the NIH (R01-AR-4676402, R01-
HL-074335, R01-AR-46588, National Center for Research Resources/
General Clinical Research Center grant M01-RR-00056, K24-AR-
02213, and P30-AR-47372), the Lupus Foundation of Pennsylvania,
the Alliance for Lupus Research, the Southeastern Pennsylvania
Chapter of the Lupus Foundation of America, and the Arthritis
Foundation.
1
Susan Manzi, MD, MPH, Natalya Danchenko, MPH: Uni-
versity of Pittsburgh School of Medicine and University of Pittsburgh
Graduate School of Public Health; 2Jeannine S. Navratil, MS, Margie
J. Ruffing, Chau-Ching Liu, MD, PhD, Sarah E. Nilson, Dale E. S.
King, Amy H. Kao, MD, MPH, Joseph M. Ahearn, MD: University of
Pittsburgh School of Medicine; 3Shanthi Krishnaswami, MBBS, MPH:
University of Pittsburgh Graduate School of Public Health, Pittsburgh,
Pennsylvania.
Address correspondence and reprint requests to Susan Manzi,
MD, MPH, S722 Biomedical Science Tower, South Wing, 3500
Terrace Street, Pittsburgh, PA 15261. E-mail: sxm6@pitt.edu.
Submitted for publication May 17, 2004; accepted in revised
form June 30, 2004.
3596
DOI 10.1002/art.20561
© 2004, American College of Rheumatology
0.001). The test was 81% sensitive and 91% specific for
SLE versus healthy controls and 72% sensitive and 79%
specific for SLE versus other diseases, and it had an
overall negative predictive value of 92%.
Conclusion. This is the first report of abnormal
levels of E-C4d in human disease. We found that
abnormally high levels of E-C4d and low levels of E-CR1
are characteristic of SLE, and combined measurement
of the 2 molecules has high diagnostic sensitivity and
specificity for lupus. Determination of E-C4d/E-CR1
levels may be a useful addition to current tests and
criteria for SLE diagnosis.
It has been known for decades that proteolytic
fragments of complement component C4, generated
during activation of the classical pathway, are present on
surfaces of normal erythrocytes. C4 is a class III HLA
molecule, and its polymorphism is responsible for the
Rogers and Chido blood group antigens; however, the
physiologic significance of erythrocyte-bound C4 (E-C4)
is entirely unknown. Although there has been extensive
study of serum C4 levels in human disease, abnormali-
ties of E-C4 and the potential role of E-C4 molecules
during inflammatory and immune responses have not
been investigated.
A large body of evidence accumulated over sev-
eral decades has demonstrated that abnormalities in
complement activation and clearance of immune com-
plexes by erythrocytes are fundamental to the pathogen-
esis of systemic lupus erythematosus (SLE) (1,2). These
include reduced levels of CR1 on erythrocytes, deficien-
cies in components of the classical pathway including C4,
and saturation of CR1 by preexisting immune complexes
(3–12). These observations led to our hypothesis that
abnormalities in complement activation specific to SLE
may be reflected by molecular events involving both
receptors and ligands on erythrocyte surfaces and that
E-C4d AND E-CR1 IN SLE
identification of such abnormal patterns may be useful in
the diagnosis of SLE.
SLE is the prototypic autoimmune disease, and
arguably one of the greatest diagnostic challenges
among rheumatologic disorders. The spectrum of dis-
ease among patients with SLE is broad and ranges from
subtle or vague symptoms to life-threatening multiorgan
failure. The manifestations of lupus often mimic those of
other diseases. Criteria were developed for disease
classification in 1971 (13) and revised in 1982 (14) and
1997 (15). These criteria are meant to ensure that study
patients from different geographic locations are compa-
rable. Of the 11 criteria, the presence of 4 or more,
either serially or simultaneously, is sufficient to classify a
patient as having SLE.
Although the criteria serve as useful reminders of
those features that distinguish lupus from other related
autoimmune diseases, they are unavoidably fallible be-
cause of the subjective nature of some criteria, the
manifestations of SLE not captured in the criteria, and
the evolving nature of the disease. There is no definitive
test for SLE, and thus, it is often misdiagnosed.
In routine practice, most physicians rely on single
or multiple serologic abnormalities to aid in the diagno-
sis of SLE. However, the most widely used laboratory
tests, such as those measuring antinuclear antibodies
(ANAs) and antibodies to double-stranded DNA
(dsDNA), have proven inadequate because of deficien-
cies in sensitivity, specificity, or both. For example, ANA
positivity is at least 95% sensitive for a diagnosis of SLE,
but it is a nonspecific serologic abnormality with a
positive predictive value of only 11% (16,17). In con-
trast, detection of high-titer antibodies to anti-dsDNA is
more than 95% specific for a diagnosis of SLE; however,
the sensitivity of this assay is low, since almost 50% of
patients with SLE have negative results on anti-dsDNA
testing (18). Ultimately, the diagnostic challenge of SLE
may result in delayed therapeutic intervention, inappro-
priate treatment of patients due to misdiagnosis, and
potential delays in and misinterpretations of clinical
trials. There is an urgent need for development of
additional diagnostic markers for SLE.
In this study, we investigated whether erythro-
cytes from patients with SLE defined by American
College of Rheumatology (ACR) criteria bear specifi-
cally abnormal levels of complement ligand C4d and its
receptor CR1. We also examined whether E-C4d/E-CR1
determinations would provide added value to testing for
ANA and anti-dsDNA in confirming a diagnosis of SLE.
3597
PATIENTS AND METHODS
Study participants. All study participants were 18
years of age or older, and all provided written informed
consent. No one was excluded based on sex or ethnicity. The
University of Pittsburgh Institutional Review Board approved
this study.
SLE patients. Consecutive patients with SLE were
recruited for this study during routine visits to the University of
Pittsburgh Lupus Diagnostic and Treatment Center. Only
patients who met the 1982 (14) or 1997 (15) ACR revised
criteria for the classification of definite SLE were recruited. As
part of their routine care, all patients underwent a history-
taking and physical examination by one physician (SM), who
was blinded to the results of tests for erythrocyte-bound
complement. To determine the spectrum of disease repre-
sented by our SLE patient sample, we obtained measurements
of disease activity at the time of the visit, using the Systemic
Lupus Activity Measure (SLAM) (19) and the Safety of
Estrogens in Lupus Erythematosus: National Assessment
(SELENA) version of the Systemic Lupus Erythematosus
Disease Activity Index (SLEDAI) (20).
Patients with other diseases. Randomly selected patients
with various other rheumatic, inflammatory/autoimmune, or
hematologic diseases (14 different diseases) were recruited.
The diagnoses were confirmed by subspecialist physicians from
various outpatient facilities at the University of Pittsburgh
Medical Center.
Healthy controls. Healthy controls were recruited
through advertisements posted on the University of Pittsburgh
campus. To confirm their healthy status, participants com-
pleted a brief questionnaire regarding obvious medical condi-
tions.
Flow cytometric characterization of erythrocytes. A
5-cc sample of blood was obtained from each study participant
at the time of the study visit. The blood sample was placed in
a Vacutainer tube (Becton Dickinson, Franklin Lakes, NJ)
containing EDTA as an anticoagulant and used for experi-
ments on the same day that it was collected. Whole blood was
diluted in phosphate buffered saline (PBS) containing 1%
bovine calf serum, and erythrocytes were pelleted, washed with
PBS containing bovine calf serum, and aliquoted for antibody
staining. Mouse monoclonal antibodies specific for human
CR1 (HB 8592; American Type Culture Collection, Rockville,
MD), human C4d (reactive with all C4d-containing fragments
of C4; Quidel, San Diego, CA), or the isotype-matched control
MOPC21 were added to erythrocytes at a concentration of 10
␮g/ml. Fluorescein isothiocyanate–conjugated goat anti-mouse
IgG F(ab⬘)2 (Jackson ImmunoResearch, West Grove, PA) was
added at a concentration of 10 ␮g/ml. Cells were analyzed by
flow cytometry using a FACSCalibur flow cytometer (Becton
Dickinson Immunocytometry Systems, San Jose, CA). Eryth-
rocytes were electronically gated based on forward and side
scatter properties to include only single cells. Surface expres-
sion of CR1 and C4d on gated cells was expressed as specific
mean fluorescence intensity (MFI) (CR1- or C4d-specific
mean fluorescence minus the isotype control mean fluores-
cence).
Statistical analysis. Descriptive statistics, including
means, medians, standard deviations, and ranges, were com-
puted for continuous data, and frequency distributions were
3598 MANZI ET AL

Table 1. Clinical characteristics of the 100 study patients with systemic lupus erythematosus*
Age, mean ⫾ SD (range) years 42 ⫾ 11.92 (18–70)
White, % 85
Female, % 94
Disease duration, mean ⫾ SD (range) years 11.0 ⫾ 8.13 (0.01–36.1)
Malar rash, % 59
Discoid rash, % 14
Photosensitivity, % 58
Oral ulcers, % 54
Arthritis, % 91
Serositis, % 56
Renal disease, % 28
Neurologic disease, % 11
Hematologic manifestations, % 68
Hemolytic anemia 19
Leukopenia 48
Thrombocytopenia 25
Immunologic test result ever positive, % 93
Anti-Sm 14
Antiphospholipid antibodies (n ⫽ 95)† 61
Antinuclear antibodies 99
Anti-SSA or anti-SSB 33
Rheumatoid factor 9
Anti–double-stranded DNA 82
Raynaud’s phenomenon 37
SLAM score, mean ⫾ SD (range) 6.00 ⫾ 4.11 (0–19)
SELENA-SLEDAI score, mean ⫾ SD (range) 2.80 ⫾ 2.15 (0–8)
Reduced serum C3 level, % (n ⫽ 99) 60
Reduced serum C4 (level, % (n ⫽ 99) 61
Erythrocyte sedimentation rate, mean ⫾ SD (range) 27.0 ⫾ 26.25 (0–122)
mm/hour (n ⫽ 98)

* SLAM ⫽ Systemic Lupus Activity Measure; SELENA-SLEDAI ⫽ Safety of Estrogens in Lupus

Erythematosus: National Assessment version of the Systemic Lupus Erythematosus Disease Activity
Index.
† Includes abnormal levels of anticardiolipin antibodies (52 patients), a positive test result for lupus
anticoagulant (11 patients), or a false-positive test result for syphilis (8 patients).

determined for categorical variables. To determine the stabil-
ity of E-C4d or its receptor (E-CR1) on normal erythrocytes,
we used erythrocytes from healthy controls, obtained at mul-
tiple visits. Repeated-measures of analysis of variance was
used to evaluate the variability of E-C4d and E-CR1 levels
over time. Spearman’s correlations were used to examine the
effect of age on C4d and CR1 levels in healthy erythrocytes.
Wilcoxon’s rank sum tests were used to determine specific
associations of E-C4d and E-CR1 with each group of partici-
pants (patients with SLE, healthy controls, and patients with
other diseases). The statistical significance of the various
results was assessed with 2-sided hypothesis testing using SAS
6.12 for Windows (SAS, Cary, NC). CART (Classification And
Regression Tree, version 4.0; Salford Systems, San Diego CA),
a nonparametric analysis, was conducted to determine the
relative importance of E-C4d and E-CR1 in distinguishing the
patients with SLE from the healthy control population and the
patients with other diseases. Cut points for E-C4d and E-CR1
levels that allowed maximum sensitivity and specificity for a
diagnosis of SLE were determined. We used the goodness-of-
split criteria applied by the Gini method (21) and arrived at a
set of rules for each of the groups in comparison with a
satisfactory level of sensitivity and specificity. We also deter-
mined the positive predictive value (the probability of having
SLE when the assay result is positive), the negative predictive
value (the probability of not having SLE when the assay result
is negative), and the diagnostic accuracy (the sum of true-
positives and true-negatives divided by the sum of all tested).
The intraclass correlation technique (22) was used to
test the reliability of the assay results between 2 technicians
using the same set of samples, reported as the interrater
reliability coefficient. To determine reproducibility of the
assay results by the same technician, the concordance correla-
tion coefficient (23) was calculated.
RESULTS
Characteristics of the study subjects. The study
population consisted of 100 patients with SLE, 84
healthy controls, and 133 patients with other diseases.
The diagnoses of the patients with other diseases were as
follows: myositis (n ⫽ 34), systemic sclerosis (n ⫽ 32),
hepatitis C (n ⫽ 18), rheumatoid arthritis (n ⫽ 16),
sickle cell anemia (n ⫽ 8), hematologic malignancies
(n ⫽ 8), primary Raynaud’s phenomenon (n ⫽ 5),
Sjögren’s syndrome (n ⫽ 3), osteoarthritis (n ⫽ 2),
E-C4d AND E-CR1 IN SLE 3599

Table 2. Results of measurement of E-C4d and E-CR1 in healthy 0.72). A broad range of disease activity, as reflected by
controls, repeated over time* the SLAM and SELENA-SLEDAI scores, and a wide
Control subject, E-C4d, E-CR1, spectrum of organ involvement were represented among
date specific MFI specific MFI the patients. Disease duration ranged from new onset to
1 longstanding and was somewhat shorter in the study
July 14, 2000 5.43 19.55 population than in the Registry population (mean ⫾ SD
July 20, 2000 5.18 18.60
January 3, 2002 5.01 11.98 11 ⫾ 8.13 years versus 15 ⫾ 7.6 years; P ⬍ 0.001). The
2 study patients were slightly younger on average than
July 13, 2000 5.95 50.83
July 14, 2000 6.18 50.06 those in the Registry (mean ⫾ SD 42 ⫾ 11.9 years versus
July 20, 2000 6.64 48.37 50 ⫾ 14.1 years; P ⬍ 0.001). Table 1 includes data on the
3 ACR criteria met and the immunologic tests for which
February 27, 2001 16.12 20.28
September 5, 2001 16.98 17.65 results had been abnormal at any time during the course
May 17, 2002 17.61 16.89 of disease. This information is available through the
4 Pittsburgh Lupus Registry. The SLAM, SELENA-
December 2, 2000 5.90 24.91
April 18, 2001 4.56 21.41 SLEDAI, C3, C4, and erythrocyte sedimentation rate
June 12, 2001 6.68 25.51 values are from the day of the study visit.
5 The healthy controls had a mean age of 37 ⫾ 12.6
April 18, 2001 2.05 34.85
May 23, 2001 3.20 32.79 years; 82% were white and 81% female. The study
June 12, 2001 3.16 38.37 participants with other diseases had a mean age of 50 ⫾
6
May 16, 2001 7.68 25.97
13.3 years; 86% were white and 68% were female.
June 20, 2001 8.52 25.76 C4d and CR1 levels on erythrocytes of healthy
March 18, 2002 5.23 23.93 controls. Demonstration of SLE-specific abnormal lev-
* E ⫽ erythrocyte; MFI ⫽ mean fluorescence intensity. els of E-C4d and E-CR1 required establishing the range
of these values in a healthy control population and
determining the stability of E-C4d and E-CR1 levels in
hemophilia (n ⫽ 2), psoriatic arthritis (n ⫽ 2), Wegen- a given individual over time. Initial studies of 84 healthy
er’s granulomatosis (n ⫽ 1), sarcoidosis (n ⫽ 1), and controls demonstrated that C4d (mean ⫾ SD specific
urticarial vasculitis (n ⫽ 1). MFI 6.7 ⫾ 5.28) and CR1 (specific MFI 22.7 ⫾ 9.32)
Demographic and clinical features of the patients could be detected on erythrocytes of all healthy controls.
with SLE are shown in Table 1. These 100 patients Stability of the E-C4d/CR1 normal phenotype
constituted a representative sample from the Pittsburgh was determined by serial studies of healthy controls.
Lupus Registry, which currently includes 1,191 living Table 2 depicts results obtained in 6 representative
participants. The race and sex composition of our study healthy controls and demonstrates the stability of the
population reflects that of the Pittsburgh Lupus Regis- E-C4d and E-CR1 levels over days, months, and years.
try: the group of participants was primarily white (85%, Thirteen healthy controls were studied during each of at
versus 82% in the Pittsburgh Lupus Registry; P ⫽ 0.50), least 3 consecutive visits. Repeated-measures of analysis
and as expected, mostly female (94% versus 93%; P ⫽ of variance on results obtained from the first 3 visits

Table 3. Comparison of E-C4d and E-CR1 levels in patients with SLE, healthy controls, and patients
with other diseases*
SLE patients Controls Patients with other diseases
(n ⫽ 100) (n ⫽ 84) (n ⫽ 133)
E-C4d, specific MFI† 24.6 ⫾ 28.53, 13.8 6.7 ⫾ 5.28, 5.7 9.28 ⫾ 6.53, 7.96
(1.94–155.03) (1.06–46.38) (0.82–52.63)
E-CR1, specific MFI† 13 ⫾ 7.16, 12.1 22.7 ⫾ 9.32, 22.2 18.43 ⫾ 7.14, 17.9
(1.41–40.89) (2.43–50.8) (3.73–38.26)

* Values are the mean ⫾ SD, median (range). SLE ⫽ systemic lupus erythematosus (see Table 2 for other
definitions).
† Median values are significantly different among the 3 groups as well as in pairwise comparison of groups
(P ⬍ 0.001).
3600
Table 4. CART analysis of the utility of E-C4d and E-CR1 levels in diagnosing SLE*
Sensitivity,
Comparison Rule†
SLE patients (n ⫽ 100) vs. C4d ⬎9.46, or
healthy controls ⫹ C4d ⱕ9.46 and CR1 ⱕ9.11
patients with other
diseases (n ⫽ 217)
SLE patients (n ⫽ 100) vs. C4d ⬎9.7, or
healthy controls (n ⫽ 84) C4d ⱕ9.7 and CR1 ⱕ6.46‡
SLE patients (n ⫽ 100) vs. C4d ⬎9.46 and CR1 ⱕ19.56‡
patients with other
diseases (n ⫽ 133)
* CART ⫽ Classification And Regression Tree; E ⫽ erythrocyte; SLE ⫽ systemic lupus erythematosus.
† Values reported as specific mean fluorescence intensity.
‡ Included to demonstrate how the specificity of the assay can be increased with slight modifications of the algorithm, depending on the diagnostic
dilemma.
indicated that, when considered either as a group or
within individual subjects, neither the levels of E-C4d
nor those of E-CR1 changed significantly over time (P ⫽
0.69 and P ⫽ 0.87 for E-C4d and E-CR1, respectively, in
the group; P ⫽ 0.61 and P ⫽ 0.86 in individual subjects).
These data demonstrate the striking stability of E-C4d
and E-CR1 in healthy individuals and suggest that levels
outside this normal range may reflect complement acti-
vation and a pathophysiologic state. We also found that
there was no effect of age on the levels of either C4d
(r ⫽ 0.04, P ⫽ 0.74) or CR1 (r ⫽ -0.16, P ⫽ 0.14) on
erythrocytes of healthy controls.
This assay was also shown to be highly reliable
and reproducible. Two technicians performed the assay
on the same set of 10 random samples, and the intraclass
correlation coefficient was 0.96 for E-C4d and 0.99 for
E-CR1. One of the technicians repeated the assay on the
same set of 10 samples, and the concordance correlation
coefficient was 0.95 for E-C4d and 0.99 for E-CR1.
Sensitivity and specificity of E-C4d and E-CR1
levels for SLE diagnosis. E-C4d– and E-CR1–specific
MFI values were then compiled for the entire study
population (patients with SLE, healthy controls, and
patients with diseases other than SLE), using blood
samples obtained at the study visit. The median values
were significantly different among the 3 groups and in
pairwise comparisons (P ⱕ 0.001) (Table 3). The SLE
patients exhibited a pattern of higher levels of E-C4d
and lower levels of E-CR1 than those found in the
healthy controls or patients with other diseases. We used
CART analysis to assess the utility of E-C4d and E-CR1
in diagnosing SLE. Using the algorithm C4d (MFI)
⬎9.46, or C4d ⱕ9.46 and CR1 ⱕ9.11, we could distin-
guish patients with SLE (n ⫽ 100) from patients with
MANZI ET AL
Positive Negative Diagnostic
Specificity, predictive predictive accuracy,
% % value, % value, % %
86 71 58 92 76
81 91 91 80 85
72 79 72 79 76
other diseases and healthy controls combined (n ⫽ 217)
with a sensitivity of 86%, a specificity of 71%, a positive
predictive value of 58%, a negative predictive value of
92%, and an accuracy of 76% (Table 4). In Figures 1A
and B, the lines on the graphs represent the cut points
for C4d (9.46) and CR1 (9.11) generated by the CART
analysis. Figure 1A illustrates the “lupus pattern” of high
C4d and low CR1 levels, and Figure 1B illustrates the
pattern observed in patients with other diseases and in
healthy controls.
In a clinical practice setting, this algorithm could
be refined to improve the specificity of the test depend-
ing on the diagnostic dilemma. For example, when SLE
patients were compared with healthy controls only, the
specificity increased from 71% to 91% by using a slightly
different algorithm, C4d (MFI) ⬎9.7, or C4d ⱕ9.7 and
CR1 ⱕ6.46 (Table 4 and Figures 1C and D). Similarly,
when SLE patients were compared with patients with
other diseases, the specificity increased from 71% to
79% with another algorithm, C4d ⬎9.46 and CR1
ⱕ19.56 (Table 4 and Figures 1E and F).
We next sought to determine whether our assay
would provide added value to testing for ANAs and
anti-dsDNA antibodies by identifying additional SLE
patients who might have negative results on tests for
ANA and/or anti-dsDNA antibody at the time of the
visit. Our cohort represented a broad range of disease
duration (0.01–36.1 years) (Table 1), and the capacity
for a diagnostic assay to identify SLE patients during a
single clinic visit throughout the disease course would be
advantageous. On the day of the study visit, 99 of 100
SLE patients were positive for ANA. Of the 99 ANA-
positive patients, 47 were positive and 51 were negative
for anti-dsDNA antibody (results unavailable in 1 pa-
E-C4d AND E-CR1 IN SLE
Figure 1. Detection of C4d and CR1 on erythrocytes (E) from
patients with systemic lupus erythematosus (SLE), patients with other
diseases, and healthy controls. Erythrocytes from patients with SLE
show a pattern of C4d deposition and CR1 expression that is distinct
from that in healthy controls or patients with other diseases. CR1-
specific mean fluorescence intensity is shown on the x-axis, and
C4d-specific mean fluorescence intensity on the y-axis, for erythrocytes
from 100 patients with SLE (A, C, and E) compared with 84 healthy
controls plus 133 patients with other diseases (B), healthy controls
alone (D), and patients with other diseases alone (F). Cut points
determined by CART analysis (Classification And Regression Tree;
see Patients and Methods) are indicated by solid lines. Gray areas
represent values of E-C4d/CR1 corresponding to a diagnosis of SLE.
tient) (Figure 2A). Of the 51 patients who were negative,
40 were positive for SLE by the E-C4d/E-CR1 assay. In
addition, the ANA-negative patient was positive for SLE
by our assay. Thus, the anti-dsDNA test alone would
have correctly identified 47 of the 100 SLE patients
(sensitivity 47%). Our assay alone identified 86 of the
100 patients with SLE (sensitivity 86%). By combining
our assay with the anti-dsDNA test, we were able to
correctly identify 89 of the 100 patients with SLE
3601
(sensitivity 89%). Furthermore, of the 51 patients who
were negative for anti-dsDNA antibody on the day of the
visit, only 28 had abnormal levels of serum C4 with or
without abnormal levels of serum C3, indicating that
measurement of E-C4d was more sensitive than mea-
surement of serum C4 in confirming a diagnosis of SLE
(Figure 2B).
DISCUSSION
This is the first report of abnormal levels of
erythrocyte-bound C4 in human disease. We have shown
that increased levels of E-C4 and decreased levels of
E-CR1 are characteristic of SLE. Combined measure-
ment of the 2 molecules is a highly sensitive and specific
approach to the diagnosis of lupus.
The current standard approach to the diagnosis
of SLE relies on the revised ACR criteria published in
1982 (14). Initial validation of these criteria demon-
strated a sensitivity of 83% and specificity of 89%.
However, it is important to note that these criteria were
actually not generated for diagnostic purposes, and they
were developed and validated using preexisting data-
bases. In routine clinical practice, use of the ACR
classification criteria for diagnosing SLE is problematic
for reasons described above. In 1997, the criteria were
again revised (15) to substitute an outdated laboratory
test (lupus erythematosus cell preparation) with a newer
laboratory test (assay for antiphospholipid antibodies,
which are frequently present in patients with SLE).
Ultimately, the goal is to identify better diagnostic and
disease-monitoring biomarkers for SLE that will elimi-
nate the need for more subjective criteria that are often
misinterpreted. The objective of this study was to iden-
tify a biomarker with sufficient sensitivity and specificity
for SLE defined by the current ACR criteria. Our next
step will be to examine whether this biomarker can
accurately identify patients who do not meet the ACR
criteria but are believed by expert opinion to have a
diagnosis of SLE. A biomarker with this potential appli-
cation would have a dynamic impact on earlier and more
accurate diagnosis by all physicians, regardless of their
expertise in SLE.
The single most useful laboratory test to date for
confirming a diagnosis of SLE has been determination
of anti-dsDNA antibodies. This is a highly specific test.
Healthy individuals essentially never produce this auto-
antibody, and it is detected in ⬍5% of patients with
other diseases. However, the mean sensitivity of anti-
dsDNA testing for SLE among published studies is only
57.3% (17). Furthermore, statistical measures of the
3602 MANZI ET AL

Figure 2. Comparison of antinuclear antibody (ANA), double-stranded DNA (dsDNA) antibody, E-C4d/CR1, and serum C4 test results. Tests for
dsDNA antibodies, serum C3 and C4, and the E-C4d/CR1 assay were performed on samples obtained on the day of the study visit. Of the 100
patients with SLE in this study, 99 were positive for ANA. Of these, 47 patients were positive and 51 were negative for dsDNA antibodies; results
were unavailable in 1 patient. A, Forty of the 51 patients who were negative for dsDNA antibodies had the E-C4d/CR1 staining pattern, consistent
with a diagnosis of SLE by the cut points shown in Table 4 and Figure 1. We were unable to obtain results of the dsDNA assay from the day of the
study visit in 1 of the 99 patients who were positive for ANA (ⴱ). This patient and the 1 patient who was negative for ANA were both positive for
SLE by the E-C4d/CR1 assay. The E-C4d/CR1 assay identified 86 of 100 patients with SLE, the dsDNA antibody test identified 47 of 100, and the
combination of the 2 tests identified 89 of 100. B, Of the 51 patients who were negative for dsDNA antibodies, 28 had abnormal levels of serum C4
(with or without abnormal levels of serum C3). Of the 47 who were positive for dsDNA antibodies, 31 had abnormal levels of serum C4 (with or
without abnormal levels of serum C3). Serum C3 and C4 results were unavailable in 1 patient (the same patient for whom dsDNA antibody results
were unavailable) (ⴱⴱ). See Figure 1 for other definitions.
E-C4d AND E-CR1 IN SLE
sensitivity of anti-dsDNA testing for the diagnosis of
SLE are usually calculated based on at least 1 positive
test result among repeated assays, with intervals from
initial physician encounter to a positive result that can
range over months, years, and even decades. For exam-
ple, in the current study, 82% of the patients with SLE
were anti-dsDNA positive at some point in the disease
course (Table 1); however, only 47% were positive at the
time of the study visit (Figure 2), as compared with 86%
who had an abnormal E-C4d/CR1 level at that visit
(Figure 2). This finding reflects the tendency of anti-
dsDNA titers to fluctuate between positive (abnormal)
and negative (normal) during the disease course. Our
data suggest that if E-C4d/CR1 values fluctuate, they are
more likely than anti-dsDNA to vary within the “abnor-
mal” range. This characteristic of E-C4d/CR1 suggests
that it may have significant utility in standard medical
practice, where it is not uncommon for a patient with
SLE to be encountered only once by a given physician,
who may be a generalist or subspecialist.
We found that, whereas an elevated C4d level
(MFI ⬎9.46) alone is sufficient to distinguish patients
with SLE from those with other diseases and from
healthy controls, the diagnosis of SLE in those with
normal E-C4d levels (ⱕ9.46) requires simultaneous
demonstration of an abnormally low level of E-CR1
(MFI ⱕ9.11). Although the explanation for this obser-
vation will require further study, it may be related to a
function of CR1. CR1 serves as a cofactor for factor
I–mediated generation of C4-derived ligands, such as
C4d (24). If participation of CR1 is necessary for
deposition of C4d on erythrocytes, abnormally low levels
of the receptor may prevent accumulation of elevated
levels of E-C4d.
In conclusion, we have developed a simple test
with high sensitivity and specificity for SLE, based on
simultaneous determination of E-C4d and E-CR1 levels
by indirect immunofluorescence and flow cytometry.
Consideration of E-C4d/CR1 levels may have significant
impact on the accuracy and timing of diagnosis of SLE in
general clinical practice, and may lead to earlier and
more appropriate therapeutic intervention.
ACKNOWLEDGMENTS
The authors are grateful to Drs. Dana Ascherman,
Brian Berk, Timothy Carlos, Albert Donnenberg, Thomas
Medsger, Chester Oddis, Margaret Ragni, William Ridgway,
and Mary Chester Wasko for providing patient blood samples
and clinical information for this study. We also thank Dr.
Janice Sabatine for manuscript preparation and editorial as-
3603
sistance, Lynne Welshons for administrative support of the
study, and Jason Brickner for preparation of graphics.
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