Measurement of Complement Components in Systemic
Lupus Erythematosus by Radial Immunodiffusion
ROBERT FINBERG, M.D., PETER BARLAND, M.D., AND ARTHUR I. GRAYZEL, M.D.
From the Division of Rheumatology, Department of
Medicine, Montefiore Hospital and Medical Center,
Bronx, New York, and the Albert Einstein
College of Medicine
Finberg, Robert, Barland, Peter, and Grayzel, Arthur I.:
Measurement of complement components in systemic lupus
erythematosus by radial immunodiffusion. Am J Clin Pathol
67: 97-99, 1977. Total hemolytic complement activity
(CH50) and levels of C4, C5, and Factor B determined by
commercially available radial immunodiffusion plates were
compared in sera from patients with systemic lupus
erythematosus. There was a significant correlation between
CH50, C4, and CS, but no correlation between Factor B and
any of the other indices of complement activity measured.
(Key words: Systemic lupus erythematosus; Hemolytic complement; C4 complement component; C5 complement component; C3 proactivator; Radial immunodiffusion; Factor B.)
pathway of complement activation, and total hemolytic
complement activity in sera from patients with SLE.
Patients
Nine patients who had active systemic lupus erythematosus documented by history and positive lupus
erythematosus cell tests, fluorescent antinuclear
antibodies, high anti-DNA titers and, in most cases,
histologic and immunofluorescent evidence of lupus
nephritis were involved in the study. Serial complement determinations were performed on sera obtained
during hospitalization or visits to the Lupus Clinic.
THE TOTAL hemolytic complement activity in serum
is considered to be a helpful measure of disease activity in systemic lupus erythematosus. Since the advent of radial immunodiffusion technics as described by
Mancini 5 and Fahey, 2 it has become cheaper and
easier to measure individual components of the complement system than to measure the total hemolytic
activity. In addition, depression of late complement
components in the presence of normal levels of early
components could be expected to indicate utilization
of the alternate pathway should this be clinically
relevant.
The purpose of this study was to determine the
correlations between C4, an early complement component utilized exclusively in the classic pathway of
complement activation, C5, a late complement component involved in both classic and alternate pathways
of complement activation, Factor B (C3 proactivator,
B2-glycoprotein II), a component of the alternate
Methods
Determinations of C4, C5 and Factor B were performed by radial immunodiffusion on the same serum
specimens. C4 and Factor B levels were determined by
radial immunodiffusion on plates manufactured by
Behring Diagnostics, Somerville, N. J. C5 levels were
determined by radial immunodiffusion on plates made
by Meloy Laboratories, Springfield, Va. The total
hemolytic complement was measured by the method of
Kent and Fife. 3 All sera were used within an hour of
collection or frozen at - 7 0 F. Correlation coefficients
were determined by linear regression analysis.
Received August 4, 1975: received revised manuscript January 7,
1976; accepted for publication March 2, 1976.
Supported by grants from the Lupus Foundation. New York
Chapter, and the Arthritis Foundation.
Dr. Finberg's present address is the Department of Medicine,
N. Y. U. Medical Center, New York, New York.
Address reprint requests to Dr. Barland: Department of Medicine,
Montefiore Hospital and Medical Center, 111 East 210th St., Bronx.
New York 10467.
Results
The reproducibilities of all three radial immunodiffusion assays were very high, with standard deviations
of duplicate specimens of less than 5%. Table 1 indicates the correlation coefficients observed when the
three radial immunodiffusion determinations were
97
I
FIN B E R G . B A R L A N D A N D G R A Y Z E L
98
—
A.J.C.P. . January 1977
25 -
E
8
- .1
r
8
-
6
i3
FIG. 1. The levels of C4 (x) and C5 (•) as
determined by radial immunodiffusion plotted
against the total hemolytic complement activity
(CH50) in serum specimens from patients with
active systemic lupus erythematosus.
X
i—i—*i—i—i—r
70
40
60
80
1
100
110
140
CH,o Uniti
1
1
1
1
1
160
110
300
220
240
T"
260
/ml
compared with the values obtained by the total hemolytic assay and when C4 was compared with C5 in the
same sera. There was a highly significant correlation
between serum C4 and C5 complement components
and between each of these components and the total
serum hemolytic complement activity. No correlation
between Factor B levels and total hemolytic complement activity or C4 or C5 was found. Figure 1
graphically depicts the correlation of C4 and C5 with
total hemolytic complement. There was no patient who
had a normal C4 value in the presence of a low C5 value,
which might have indicated pure alternate pathway
activation. In fact, the number of abnormally low (less
than 20 mg/100 ml) C4 values obtained was greater than
the number of low C5 values (less than 5 mg/100 ml).
Discussion
The commercially available radial immunodiffusion
assays for the third component of complement show
Table I. Regression Coefficients (R) and p Values
for Various Assays of Complement Activity
Variables
No. of Samples
R
P
C5 and CH50
C4 and CH50
Factor B and CH50
Factor B and C5
Factor B and C4
C5 and C4
27
26
27
31
30
30
.794
.728
.141
.100
.000
.751
<.01
<.01
>.5
>.5
>.5
<.01
considerable variability because of technical problems
related to antigenic conversion in vitro. As pointed out
by Davis and associates,1 Vladutiu,8 and Perrin,6 values
obtained by these measurements vary with the type of
container, the storage temperature, and the time of
collection. We have observed similar results in sera
from patients with systemic lupus erythematosus.
Thus, this method is not a reliable indicator of complement activity. Our data indicate that should a laboratory, for reasons of economy and time, seek a substitute
for the measurement of total hemolytic complement
activity in order to follow patients with lupus glomerulonephritis, measuring C4 levels by radial immunodiffusion is probably most satisfactory.
By measuring components on either side of the alternate pathway shunt, we had hoped to find instances of
pure alternate pathway utilization as reflected by low
C5, Factor B and total hemolytic complement levels in
the presence of normal C4. That no such group was
found and that there was a highly significant regression
coefficient between C4 and C5 support the conclusion
that in the great majority of cases of systemic lupus
erythematosus the low complement levels observed are
the result of the classic pathway activity with or without
the involvement of the "amplification loop." 7
It is presently known that the classic pathway is
involved in systemic lupus erythematosus but the relative importance of the alternate pathway has not been
completely assessed. Hunsicker and associates4 found
COMPLEMENT DETERMINATIONS IN SLE
Vol. 67 • No. I
a decrease in Factor B in association with intense
disease activity, but they were uncertain whether
similar changes were present at other times. Since we
have found no correlation between Factor B levels and
either total hemolytic complement levels or the level of
C5, we would question whether the level of Factor B as
determined by radial immunodiffusion is an accurate
reflection of alternate pathway activity.
References
1. Davis NC, West CD, Ho M: Effect of aging in serum on quantitation of complement component C3. Clin Chem 18:1484-1487,
1972
99
2. Fahey JL, McKelvey EM: Quantitative determinations of serum
immunoglobulins in antibody-agar plates. J Immunol 94:
84-90, 1965
3. Hook WA, Muschel LH: Anticomplementary effects and
complement activity of human sera. Proc Soc Exp Biol Med
117:292-297, 1964
4. Hunsicker LG, Ruddy S, Carpenter CB, et al: Metabolism of
third complement component (C3) in nephritis. N Engl J Med
287:835-840, 1972
5. Mancini G, Carbonara A, Heremans J, et al: Immunochemical
quantitation of antigen by single radial immunodiffusion.
Int J Immunochem 2:235-254. 1965
6. Perrin J: C3 Standards. Lancet 1:1189, 1975
7. Ruddy S: Chemistry and biological activity of the complement
system. Transplant Proc 6:1-7, 1974
8. Vledutiu A: C3 Standards? Lancet 1:979, 1975