To the Editor.—The number of patients with negative antinuclear antibodies (ANAs) and positive extractable nuclear antigen antibodies (ENAs) appears to be greater than we expected. However, medical colleges1  and insurance companies support the performance of ENA testing only in patients with positive ANAs, or in a few cases with suspicions of Sjögren syndrome, suspicions of myositis, or suspicions of systemic lupus erythematosus (SLE) with ANA negativity, and algorithms that consider this criterion have been designed to optimize ENA indications.2  This study was proposed to evaluate the frequency of cases with negative ANAs and positive ENAs in a cohort of 2510 patients with suspected systemic autoimmune disease of the connective tissue.

Tests were run between January 1999 and December 2014 in a private clinical laboratory by the same technician. The requests were made by any doctor who suspected a systemic autoimmune disease. By indirect immunofluorescence microscopy, ANA and anti-centromere antibodies were made using Hep2 cells (DiaSorin, Saluggia, Italy) as a substrate starting from a 1:80 dilution. Hep2 ANA tests were performed and interpreted following the American College of Rheumatology (ACR) recommendations.

Using enzyme-linked immunosorbent assay, antibodies were determined against the following extractable nuclear antigens (1:100 dilution): Sm, RNP, Jo-1, Scl-70, SSA (Ro), and SSB (La) (DiaSorin).

Among the 2510 ENA studies performed in combination with ANAs, 2128 (84.8%) were performed in women and 382 (15.2%) in men. We found that 86 of the 2510 cases (3.4%) were negative to ANAs but positive against at least 1 antigenic subspecificity of ENA, distributed as follows: 47 with only anti-SSA, 8 only with anti-SSB, 5 with anti-SSA and anti-SSB, 1 with anti-SSB and anti-RNP, 7 with only anti-RNP, 1 with only anti-RNP, 3 with anti–Scl-70, 2 with anti–Scl-70 and anti-SSA, 1 with anti–Scl-70 and anti-SSB, 5 with anti–Jo-1, and 4 with anti-centromere.

The association of ANAs and ENAs with each other and with the sexes is shown in the Table. As we expected, the strength of the association was more relevant in men with ANA-negative, ENA-negative antibodies and in women with ANA-positive, ENA-positive antibodies (χ2 = 33.689; degrees of freedom = 3; P < .001).

Antinuclear Antibodies (ANAs) and Extractable Nuclear Antigen Antibodies (ENAs) According to Sexa

Antinuclear Antibodies (ANAs) and Extractable Nuclear Antigen Antibodies (ENAs) According to Sexa
Antinuclear Antibodies (ANAs) and Extractable Nuclear Antigen Antibodies (ENAs) According to Sexa

The ANA-negative, anti-ENA–positive group represented 3.4% of all patients (86 of 2510), and 91.86% (79 of 86) of this subgroup was represented by women. The number of patients does not seem to correspond in all cases to some positive forms of anti–Jo-1 myositis and negative ANA, or to cases with SLE or Sjögren syndrome with positive SSA and negative ANA3  because the frequency of these conditions is low. For example, it has been estimated that approximately 3% of individuals with SLE who are ANA negative may be positive for antibodies against cardiolipin, anti–double-stranded DNA, or anti-SSA/Ro.4  Most Ro-positive patients show a fine speckled pattern where the resting cells show fine or diffuse speckling throughout the nuclei (but not the mitotic cells), and it is often due to Ro and La antibodies.5  The 1:40 ANA tests were done on those discrepant cases, searching if the Hep2 substrate displayed cytoplasmic fluorescence for those cases in which the nuclei were negative. From 47 sera ANA-negative and just Ro-positive patients, 26 had cytoplasmic staining, which may suggest the presence of antibodies to ENA in the absence of nuclear staining. The expression of Ro antigens in the cytoplasm or nucleus is linked to the stage of the cell cycle. When the study was conducted, it was not possible to prove if weakly positive ENA sera were caused in some cases by substantial antibody titers that react with antigens on particular ENA components, such as Ro52 or Ro60, that may have been poorly conserved in the cells of the Hep2 substrate.

On the one hand, it is possible that some antibodies directed to extractable nuclear antigens are part of the normal immune response, and on the other hand, in this subgroup there may be individuals at risk for developing autoimmune diseases. In some patients with SLE and Sjögren syndrome, ANAs have appeared even years before the first symptoms appear.6 

Even when choosing wisely3  and when ACR recommendations that suggest an ANA screen before an ENA test is performed are widely accepted, it is striking that among 732 patients positive for at least 1 of the 2 groups of autoantibodies (ANA and ENA), 86 (11.75%) were positive for at least 1 ENA but none for ANA. The first and only serologic marker of autoimmunity detectable in this subgroup of patients was 1 or more ENA antibodies, and we do not know what this implies even when we accept the absence of clinical correlation as a potential limitation of this study.

Searching Hep2 substrate cytoplasmic fluorescence for those cases in which the nuclei were negative and a proper use of methods of detection of substantial antibody titers that react on particular ENA components (such as fluorescence enzyme-linked immunosorbent assay, human recombinant Ro52, Ro60 proteins, and immunoglobulin G antibodies when ENA screens are positive in accordance with the National Health Service South Tees Hospitals criteria7 ) are just some strategies to employ on the discrepant cases.

We would like to acknowledge our great friend Carlos Béjar Lozano, BSc, author of this work, who recently passed away from COVID-19 infection, and who promoted the development of the clinical laboratory and, in particular, of immunology in our region.

1.
Davis
LA
,
Goldstein
B
,
Tran
V
, et al.
Applying choosing wisely: antinuclear antibody (ANA) and sub-serology testing in a safety net hospital system
.
Open Rheumatol J
.
2015
;
9
:
82
87
.
2.
Man
A
,
Shojania
K
,
Phoon
C
, et al.
An evaluation of autoimmune antibody testing patterns in a Canadian health region and an evaluation of a laboratory algorithm aimed at reducing unnecessary testing
.
Clin Rheumatol
.
2013
;
32
(5)
:
601
608
.
3.
Yazdany
J
,
Schmajuk
G
,
Robbins
M
, et al.
Choosing wisely: the American College of Rheumatology's Top 5 list of things physicians and patients should question
.
Arth Care Res
.
2013
;
65
(3)
:
329
339
.
4.
Alarcon-Torres
I
,
Gonzalez-Rodriguez
C
,
Jimenez-Jimenez
J
,
Fernandez-Suarez
A
,
Alsina-Donadeu
M.
Actualización en el manejo de los anticuerpos antinucleares en las enfermedades autoinmunes sistémicas: recomendación (2014)
.
2021
.
5.
Buchner
C
,
Bryant
C
,
Eslami
A
,
Lakos
G.
Anti-nuclear antibody screening using HEp-2 cells
.
JoVE
.
2014
;
(88)
:
e51211.
6.
Grygiel-Górniak
B
,
Rogacka
N
,
Puszczewicz
M.
Antinuclear antibodies in healthy people and non-rheumatic diseases–diagnostic and clinical implications
.
Reumatologia
.
2018
;
56
(4)
:
243
248
.
7.
Anti-Ro antibodies
.
NHS South Tees Hospitals Web site
.
October
6,
2021
.