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Incidence of antinuclear antibodies in Japanese patients with chronic fatigue syndrome.

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2095
LETTERS
disease activity in systemic lupus erythematosus: review. Lupus
423-94. 1995
Use of heat inactivation in assays for antibodies to
P,-glycoprotein I and anticardiolipin: comment on the
concise communication by Roubey et a1
To the Editor:
We read with interest the communication by Roubey
and colleagues (1) in which, from their analysis, the authors
suggest that the enzyme-linked immunosorbent assay (ELISA)
for antibodies against p,-glycoprotein I (P,GPI), as compared
with a conventional anticardiolipin antibody (aCL) assay, has
greater specificity for clinical manifestations of the antiphospholipid (aPL) syndrome. Among other results, Roubey et a1
describe 3 patients whose sera were positive in the anti-P,GPI
ELISA, but negative in the aCL assay. Clinical features of the
aPL syndrome were identified in 2 of those 3 patients. The
authors suggest that clinically important autoantibodies to
human &GPT may not be detected in standard aCL assays in
certain instances, and they list 2 possible explanations. First,
the anti-P,GPI antibodies in these patients' sera may be
species-specific, recognizing human, but not bovine, &GPI.
Second, thesc anti-&GPI antibodies may be directed exclusively against epitopes that are not exposed when &GPI is
bound to a phospholipid-coated surface.
We wish to draw attention to a third explanation not
mentioned by Roubey et al. W e have shown that one source of
variability in conventional aCL assays is related to the bovine
serum used for the sample dilution and blocking steps (2).
When we tested serum samples from primary aPL syndrome
patients for aCL titers in conventional ELISAs using either
heat-inactivated (WC, 30 minutes) or unheated bovine serum,
a significantly lower titer of aCL antibodies was shown in the
ELISA in which heat-inactivated bovine serum was used. This
finding may contribute to an explanation of the discordant
immunoassay results in some sera that are positive for anti-p,
GPI and appear to be negative for aCL antibodies. To explain
this, we have hypothesized that heat inactivation may induce
more changes in the antigenicity of &GPI (3) than in its
interaction with cardiolipin (4), and in addition, as described
with normal human sera and some primary aPL syndrome
patient sera (5,6), aCL antibodies and anti-P,GPI antibodies
may become unmasked after heat inactivation of bovine serum
and may block the antigenic sites before addition of human
serum samples.
Roubey et a1 used calf serum in their ELISA. In their
report, and in future reports comparing aCL and anti-P,GPI
antibodies, it would be useful to know if ELISAs were performed using heat-inactivated or non-heat-inactivated bovine
serum.
Arnulfo Nava, MD, MSc
H6pital Notre-Dame
Montreal, Quebec, Canada
Pedro A. Reyes, M U
Instituto Nacional de Cardiologia
Mkxico City, Mexico
1. Roubey RAS. Maldonado MA, Byrd SN: Comparison of an
enzymc-linked immunosorbent assay for antibodies to &glycoprotein I and a conventional anticardiolipin immuno
Arthritis Rheum 391606-1607, 1996
2. Nava A, Bafiales JL, Reyes PA: Heat inactivation of bovine serum
used for blockade in immunoenzymatic assay is associated with
spurious fall on the titers of anticardiolipin antibodies in primary
antiphospholipid syndrome sera. J Clin Lab Anal 7:116-118, 1993
3. McIntyre JA, Wagenknecht DR, Triplett DA: Detection of antiphospholipid antibodies in heat inactivated normal human sera
(lettcr). Thromb Res 69:489-490, 1993
4. Kertesz Z, Yu B-B, SteinkassererA, Haupt H, Benham A, Sim RB:
Characterization of binding of human p,-glycoprotein I to cardiolipin. Biochem J 310315-321, 1995
5. Nava A, Bafiales JL, Reyes PA: Effect of heat inactivation and
sheep erythrocyte adsorption on the titer of anticardiolipin antibodies in primary antiphospholipid syndrome and healthy blood donors
sera. J Clin Lab Anal 6:148-150, 1992
6. Cabiedcs J, Cabral AR, Alarcon-Segovia D: Identification of four
subpopulations of IgG anticardiolipin antibodies in patients with
primary antiphospholipid syndrome o n the basis of their requirement for P2-glycoproteinI and their unmasking by heat. Clin Exp
Rheumatol 12:123-127, 1994
Reply
To the Editor:
In our study comparing anti-&GPI and conventional
aCL ELISAs, bovine serum in the blocking agent and sample
diluent of the aCL assay was not heat inactivated.
Robert A. S. Roubey, M D
University of North Carolina
Chapel Hill, NC
Incidence of antinuclear antibodies in Japanese
patients with chronic fatigue syndrome
To the Editor:
W e read with interest the report by von Mikccz et al,
describing autoantibodies in patients with chronic fatigue
syndrome (CFS) (1). In 1995, we reported on the incidence of
antinuclear antibodies (ANA) in Japanese patients with CFS
( 2 ) . Our findings had certain similarities to, and certain
differences from, the results reported by von Mikecz et al.
Between 1991 and 1994, a total of 96 Japanese patients
at the outpatient clinic of the department of internal medicine
of our hospital were diagnosed as having CFS (2) according to
the diagnostic criteria of Holmes et a1 (3). W e studied these
patients as well as a control group of 49 age- and sex-matched
healthy subjects. ANA were evaluated by the standard indirect
immunofluorescence technique using HEp-2 cells produced in
Japan (Medical and Biological Laboratories, Nagoya, Japan)
as substrate (4). Each specimen was examined independently
by each of the authors, under blinded conditions. Patients were
considered to be positive for ANA if immunofluorescent
staining was observed at a serum dilution of ?1:40. Precipitating autoantibodies in the patients with CFS were examined by
double immunodiffusion (4).
Of the 96 Japanese patients with CFS, 24 (25%) were
positive for ANA, whereas only 3 of the 49 healthy subjects
(6%) were positive (P,,,,,
= 0.0112 by chi-square test) (2). The
LETTERS
2096
from a manufacturer in Japan (Medical and Biological Laboratories). Accordingly, if thc conclusions reported by von
Mikecz and colleagues were based on the high frequency of the
nuclear envelope pattern in patients with CFS, results in
controls such as patients with R A should also have been
presented.
There is no standard approach to identifjiing patients
with CFS. Additional detailed studies o n ANA in these
patients may improve our understanding of the underlying
immune disorder and the optimal method of diagnosis.
Masahiko Nishikai, MD
Satoshi Kosaka, BS
The Second Tohyo National Hospital
Tohyo, Japan
Figure 1. Representative antinuclear antibody pattern on immunofluorexence microscopy, in a Japanese patient with chronic fatigue
syndrome.
highest ANA titer was 1320. The staining pattern was speckled
in 80% of the ANA-positive patients (Figure 1). The nuclear
envelope staining pattern was found in only 1 of the 49 healthy
subjects and none of the CFS patients. Only 5 CFS patients
exhibited positive precipitating antibodies (not significantly
different from the finding in our healthy control subjects [ 1 of
49; 2761). Interestingly, 4 of the 5 patients with positive
precipitating antibodies werc negative for ANA, suggesting
that, in the patients with ANA, the partner antigens of ANA
were insoluble.
Our observations regarding the frequency of positive
ANA were similar to those in a study of CFS patients in
Seattle, Washington, reported by Bates et al (S), and suggest
the absence of a racial difference in this disorder. Relatively
low titers of ANA in CFS patients as compared with those in
lupus patients, and the insoluble nature of the partner antigens
to ANA in the CFS patients, are commonly reported in
Japanese as well as American patients with CFS (1,6).
The greatest differences between our results and those
of voii Mikecz et al involve the frequency (25% versus 68%)
and the immunofluorescent staining pattern of ANA. In 2
studies from the Scripps Research Institute (1,h) and the study
from the University of Washington (5) it appears that sera
from the same patients were used, and the same HEp-2 cells
were used as nuclear materials. Nevertheless, those studies
demonstrated widely differing frequencies of ANA positivity
(15% [5] versus 68% [1,6]). This might be due to differences in
the experience or proficiency of the technicians in each group.
Whether the authors evaluated the specimens in a blinded
manner was not specified.
It should be noted that differences in HEp-2 cells
obtained from different manufacturers can influence the frcquency or nature of staining patterns. Our previous experience
with HEp-2 cells purchased from a company in the US
(Kallestad, Chaska, MN) demonstrated a staining pattern of
the nuclear membrane type, or the nuclear envelope type, in
32% of 304 Japanese patients with rheumatoid arthritis (RA)
and in 1% of 73 healthy subjects ( 7 ) . This staining pattern was
not found in R A patients studied using HEp-2 cells obtained
1. Von Mikecz A. Konstantinov K, Buchwald DS, Gerace L, Tan EM:
Iligh frequency of autoantibodies to insoluhule cellular antigens in
patients with chronic fatigue syndrome. Arthritis Rheum 40:295305, I997
2. Nishikai M, Kosaka S: Antinuclear and anticytoplasmic autoantibodies in chronic fatigue syndrome. In, Annual Reports of Ministry
and Wclfarc: Research Committee on Epidemiology and Pathogcnesis of Chronic Fatigue Syndrome. Tokyo, Ministry of Health and
Welfare, 1995
3. Holmcs GP, Kaplan JE, Gantz NM, Komaroff AL, Schoenherger
LB, Straus SE, Jones JF, Ilubois Rk, Cunninghnm C, Rundles J,
Pahwa S, Tosato G, Zegans LS, Pultilo DT, Brown N, Shooley R,
Brus I: Chronic fatigue syndrome: a working definition. Ann Intern
Med 108:387-389, 1988
4. Nishikai M, Reichlin M: Heterogeneity of precipitating antibodies
in polymyositis and dcrmatomyositia: characterization of the Jo-1
antibody system. Arthritis Rheum 23:881-888, 1980
5. Bates DW, Buchwald D, Lee J, Kith P, Doolittle T, Rutherford C,
Churchill WH, Schur PI*, Wcner M, Wybcnga D, Winkclman J,
Komaroff AL: Clinical laboratory test findings in patients with
chronic fatigue syndrome. Arch Intern Med 155:97-103, 1995
6. Konstantinov K, von Mikecz A, Buchwald D, Jones J , Gerace L,
Tan EM: Autoantibodies to nuclear envelope antigens in chronic
fatigue syndrome. J Clin Invest 98: 1888-1896, 1996
7. Nishikai M, Sato A: Antinuclear antibodies in rheumatoid arthritis:
study by indirect immunofluorescence using HEp-2 cells as suhstrates (abstract). Ryumachi 28:654, 1988
Reply
To the Editor:
We did not cite the findings of Nishikai and Kosaka
because their 1995 data were submitted as a report to the
Ministry of Health and Welfare in Japan; such reports are not
generally available to the international scientific community.
Their studies are restricted to the use of the indirect immunofluorescence technique to determine the prevalence of ANA in
Japanese patients with CFS. In our 2 recent reports on CFS,
indirect immunofluorescence was only one of the methods
used to characterize autoantibodies (I,?). Other methods
employed in the study reported in Arthr1ti.s B Rhcurnatism
included the use of ccll and nuclear matrix preparations as
substrates, and immunoblotting with recombinant human vimentin (1). In the other study, purified rat liver nuclear
envelope preparations were used as substrate in Western
blotting, and in vitro translation product of a complementary
DNA encoding human lamin B1 was used in immunoprccipi-
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incidence, japanese, patients, antibodies, syndrome, antinuclear, fatigue, chronic
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