Key words cytoplasmic ANA pattern - reflex testing - anti-mitochondrial antibody - autoimmunity - anti-nuclear antibody
Schlüsselwörter Antinukleäre Antikörper - zytoplasmatisches ANA-Muster - Reflextest - antimitochondrialer Antikörper - Autoimmunität
Introduction
Determining anti-nuclear antibodies (ANA) using indirect immunofluorescence assays
(IIFA) on human epithelial cells (HEp- 2) is a “gold” standard test
for first level screening of systemic autoimmune rheumatic diseases (SARD)
[1–3]. The IIFA technique on HEp-2 cells has the advantage of added clinical
value, since it works as a “natural array” that allows the detection
of more than 30 different nuclear, cytoplasmic and mitotic cell patterns, by
presenting numerous native antigens [1, 2].
ICAP provides a recommended model of the nuclear patterns, cytoplasmic and mitotic
apparatus patterns ANA Test Report with clinically relevant content to harmonize for
a meaningful report in accordance with ISO 15,189, which is a challenge in
autoimmunity laboratories [1, 2].
The recommendations for reporting nuclear, cytoplasmic, and mitotic apparatus ANA
patterns by IIFA should be concise and traceable and that the report should consist
of a minimum of five well-defined parts: patient and referring physician
identifiers, ANA patterns (ICAP AC code and pattern descriptor), titer, reference
range, and comments or remarks [2 ]
[3 ]
[4 ].
While the nomenclature for ANA patterns has reached a consensus within the
International Consensus Of ANA Patterns (ICAP) workshops, neither the American
College of Rheumatology (ACR) nor the European Autoimmunity Standardization
Initiative/International Union of İmmunological Societies
(EASI/IUIS) recommendations state a clear position on which
cytoplasmic/mitotic apparatus patterns should be considered ANA negative or
positive [1–3, 5]. If an ANA positive reporting proposal is adopted, the
pattern name could be written under the cytoplasmic ANA subtitle [2]. Adding the
subtitle allows for many clinically important cytoplasmic patterns to be now
reported as positive, calling the necessary attention of the ordering physician to
meaningful results.
ANA testing using the IIFA technique necessitates the characterization of positive
ANA results through immunoassays for the detection of specific nuclear and
cytoplasmic autoantibodies in the framework of the recommended two-tier approach [6,
7]. There are several biological and non-biological limitations inherent to the IIFA
method. Visual evaluation is time-consuming, inter-observer variability may occur,
ad-hoc training is needed, and expert morphologists are required. In addition, the
photobleaching effect, the lack of automated procedures, low predictive value of the
ANA test, the variability of cellular substrates, the need for visual determination
of the pattern, and lack of specificity all contribute to the difficulties of this
method [1, 8–15]. The combination of HEp-2 IIFA and solid phase assays (SPA)
with the most relevant nuclear and cytoplasmic antigens has balanced the limitations
and increased the specificity [16]. The two-tier approach fits a reflex test which
is a ‘‘cascade’’ diagnostic approach where a
positive initial (first level) test automatically triggers further (second level)
tests based on predefined rules applied to information systems [7, 17, 18]. However,
the two-tier approach has proved to be laborious despite apparent advantages such as
only requiring a single visit to the doctor and laboratory, allowing for a rapid
clinical diagnosis, as well as potential economic profit resulting from a decrease
in unneceassary second-level tests [1, 7, 17–19]. Many laboratories cannot
integrate reflex tests into their testing algorithm, mostly due to a lack of
reimbursement policies. In these circumstances, the second line of testing is
advised based on the report, as is done in our country of Turkey [17].
For nuclear and cytoplasmic ANA patterns, the reflex test cascade would be different.
For nuclear ANA patterns, the reflex test cascade is mostly ENA testing (Extractable
Nuclear Antigens) (by ELISA or immunoblotting methods). For cytoplasmic patterns,
the reflex cascade is variable. For cytoplasmic reticular patterns, the reflex test
cascade is an anti-mitochondrial antibody (AMA) (by the IIFA method); for
cytoplasmic linear patterns, it is anti-smooth muscle antibodies (actin antibodies;
ASMA) (by the IIFA method) and for cytoplasmic dense fine speckled patterns, the
reflex test cascade is mostly ENA testing (Extractable Nuclear Antigens) (by ELISA
or immunoblotting methods) for anti-ribosomal P protein antibody [17].
Reflex testing is advised for all relevant ANA patterns, including cytoplasmic ones
which could be reported as ANA positive or ANA negative according to the ICAP
recommendations. The effect of different reporting layouts of cytoplasmic ANA
patterns on reflex testing is discussed in this study. We compared the number of
reflex test orders (AMA/ASMA) recommended between the positive reported
cytoplasmic reticular/linear ANA patterns and the negative ones.
Materials and Methods
Clinical Sample Selection
Fifty one (51) different primary, secondary, and tertiary healthcare centers in
all seven geographic regions of Turkey sent blood samples to our Clinical
central laboratory for ANA IIFA testing. In our single central laboratory, all
the analysis were performed. The selection of the patients, testing proc
edures and follow-up period took 5 years between 2014 and 2019.
In this study, 1241 patients who were monitored with suspicion of autoimmune
liver disease and who had positive cytoplasmic (reticular and linear fibrillar
ANA patterns) ANA patterns were included. 442 patients were reported as ANA
negative and 799 as ANA positive.
The type of assay used (IIFA), reported pattern, antibody levels and the advice
for reflex testing (noted as “AMA/ASMA reflex test was
recommended in case of suspicion of autoimmune hepatitis/biliary
disease.”) were identical for all patients, as per ICAP recommendations.
The laboratory wrote the pattern name under the cytoplasmic ANA subtitle
(
[Fig. 1 ]
) .
Fig. 1 ANA reporting algoritm.
At the reports of totally 215 (=79 from ANA negative reported
group+136 from ANA positive reported group) patients, the advice for
reflex testing note have not written, since the reflex test (AMA/ASMA) ordered
already with ANA test. For the next two years, we followed up the patients that
we recommended reflex tests at their reports for AMA/ASMA and ENA reflex
orders.
We prepared a questionare with two questions for the clinicians who
haven’t order any reflex;
1-Did you read the recommendation note at the report?
2- If answer is yes, why did you not order any reflex test?
2 i- clinically not needed
2 ii-patient stop follow-up
ANA and Reflex Testing
ANA tests were performed using the HEp-2 Standard kit for Helios automated IFA
systems (Aesku, Wendelsheim, Germany). Helios automated IFA systems were used to
capture images of the ANA slides and were added to the report through a
Laboratory Information System (LIS) and then stored (
[Fig. 1 ]
). ANA pictures could add
value, improving the report layout and moving towards a better education of the
ordering physician. Additionally, two IIFA experts (one laboratory technician
and a doctor) examined the slides blindly using a Led Microscope (Motic, Hong
Kong). In the case of non-conformity between the two readings, an ANA test was
run using the Mosaic HEP-20–10/Liver (Monkey) (Euroimmun,
Lüebeck, Germany).
AMA/ASMA tests were performed with rLKS–Rat wrapped
(Rat/Monkey) kits for Helmed IFA systems (Aesku). Slides were read by
two IIFA experts (one laboratory technician and a doctor) blindly on a Led
Microscope (Motic, Hong Kong). In the case of non-conformity between two
readings, AMA/ASMA test were run with the Euro plus LKS Mosaic
(Euroimmun).
ENA tests were performed with ANA-17 comp kits using Helmed Blot systems (Aesku).
In the case of non-conformity between the ANA and ENA results, ENA tests were
run with the Euroline ANA Profile 1 (IgG) kit (Euroimmun).
Statistical Analysis
For Statistical analysis, the IBM SPSS Statistics software Version 26 (SPSS Inc.,
Chicago, IL) was used. Pearson chi square test for categorical variables was
used for the comparison of reflex testing between the two groups. The results
were evaluated within a confidence interval of 95%, and a p value of
less than 0.05 was considered statistically significant.
Results
Demograhic analysis of 1241 are summarized at [Table
1 ]. 442 patients were reported as ANA negative and 799 as ANA positive.
% 83 of ANA negative reported group is female and %83,97 of ANA
positive. The mean of ages in years at ANA negative reported group is 47,3 and ANA
positive is 46,8.
Table 1 Demographic information of patients Abbrevation: ANA :
anti-nuclear antibody.
ANA Positive n: 799
ANA Negative n:442
Gender
Female n:671 (83,97%)
Female n:367 (83,03%)
Age, years
46,8 (15–89)
47,3 (17–90)
Analysis of AMA/ASMA Patient Testing
Numbers of AMA/ASMA tests performed were analyzed bi-annually ([Table 2 ]). AMA/ASMA test orders
were higher for cytoplasmic reticular/linear patterns reported as ANA
positive than negative (p-value<0.05). In the positive group with
recommendation note for reflex (AMA/ASMA) testing (n=663),
AMA/ASMA (n=181, 27.30%) tests were ordered. In the
negative group (n=363), 52 AMA/ASMA (n=20,
5.51%) tests were ordered
Table 2 Distribution of reflex test ordering at
cytoplasmic ANA patterns reported as positive and negative group.
Abbrevation: ANA : anti-nuclear antibody, AMA: anti-mitochondrial
antibody, ASMA: anti-smooth muscle antibody, ENA: Extractable
nuclear antigens.
Patients with positive cytoplasmic ANA patterns
ANA Positive
With recommendation note for reflex (AMA/ASMA)
testing
AMA/ASMA Reflex test ordering n:181 (27.30%)
(p-value<0.05)
n: 1241
n: 799
n: 663
ENA Reflex test ordering
n:86 (12.97%) (p-value<0.05)
No reflex test order
n:396 (59.7%3)
Without recommendation note for reflex (AMA/ASMA)
testing
n: 136
ANA Negative
With recommendation note for reflex (AMA/ASMA)
testing
AMA/ASMA Reflex test ordering
n:442
n: 363
n:20 (5.51%) (p-value<0.05)
ENA Reflex test ordering
n:4 (1.10%) (p-value<0.05)
No reflex test order
N:339 (93.39%)
Without recommendation note for reflex (AMA/ASMA)
testing
n: 79
Analysis of ENA Patient Testing
Although at the reports there was a recommendation note as
“AMA/ASMA reflex test is recommended in case of the suspicion of
autoimmune hepatitis/biliary disease,” the order numbers of ENA
tests even not mentioned at the reports was higher. ENA test orders for
cytoplasmic patterns reported as ANA positive group, were higher than those for
the ANA negative group (p-value<0.05) ([Table 2 ]). In the ANA positive group (n=663), the clinicians
ordered 86 ENA (12.97%) tests, and 4 ENA (1.10%) tests in the
ANA negative group (n=363).
Despite of the significant differences of reflex test ordering numbers, the
result distribution of AMA/ASMA and ENA reflex testing were smiliar in
both group ([Table 3 ]) (p>0.05).
Ratio of positive AMA/ASMA reflex test results was 74.03% in ANA
positive group and 75.00% in ANA negative group. Ratio of positive ENA
results was 1.16% in ANA positive group and 0.00% in ANA
negative group.
Table 3 Distribution of AMA/ASMA and ENA reflex
testing results. Abbrevation: ANA : anti-nuclear antibody, AMA:
anti-mitochondrial antibody, ASMA: anti-smooth muscle antibody, ENA:
Extractable nuclear antigens.
ANA Positive
ANA Negative
AMA/ASMA Positive
134 (74.03%)
12 (75.00%)
AMA/ASMA Negative
47 (25.97%)
4 (25.00%)
ENA Positive
1 (1.16%)
0 (0.00%)
ENA Negative
85 (98.84%)
4 (100.00%)
Patients With No Reflex Tests Ordered
In a large group of patients, any reflex test neither AMA/ASMA nor ENA
tests were performed ([Table 2 ]). To
analyze this omission, we contacted with their clinicians; however, not all
these clinicians were available for comment. Totally 20 of them filled the
questionare ([Table 4 ]).
Table 4 Distribution of the reasons why clinician do not
order reflex testing. Abbrevation: ANA : anti-nuclear
antibody.
ANA Positive n:396
ANA Negative n: 339
Not clinically needed
30.05%
12.97%
Not read the note
67.68%
82.89%
Not followed
2.27%
4.14%
The result of the questionare filled with the clinicians who didn’t order
reflex tests;
1–70 % from the positive and 85% from the negative
groups, admitted that they did not read the recommendation notes.
2–30 % from the positive and 10% from the negative
groups declared that they did not need any further tests according to the
patient’s clinical results.
3 –0% from the positive and 5% from the negative
groups said that their patients failed to report for the follow-up visits.
More than half declared that they did not read the recommendation notes. This
number was even higher in the ANA negative group (p-value<0.05);
however, positive reporting alone is not enough to understand the recommendation
notes.
Discussion
The ICAP has proposed that cytoplasmic ANA patterns can be reported as either
positive or negative. However, they recommend reflex testing (ICAP recommendation
13) to improve the utility of the serological evaluation regardless of whether ANA
is reported as positive or negative [1, 4]. Results of the ANA should be
communicated to clinicians through reflex test recommentation notes , bridging the
technical knowledge of the laboratory with the clinical relevance, and so adding
value to clinical decision-making,. Also we allocated time for additional
communication between the author and the clinician only for exceptional cases as a
consequence of our extensive sample collection network (51 different centers) and an
intensive work load (Our ANA test number per month is over 10,000). We wanted to
analyse how clinical perception, attraction, and attention impacted clinical
decision. If a report attracted the attention of the clinician, he read the whole of
the report and advised the ordering of a reflex test whenever clinically needed.
Paying attention to the advice notes is crucial in our country of Turkey, since
there is no reimbursement policy for the reflex cascade algorithm. So we compared
the recommended reflex test ordering frequency between two types of cytoplasmic
pattern reports; ANA positive and negative [1].
In our study, we found that an ANA positive cytoplasmic reticular/linear
pattern led to a higher AMA/ASMA reflex testing rate when compared to the
ANA negative group (p-value<0.05). Positive reports attracted more attention
from the clinician and, consequently, more ICAP recommended reflex tests were
ordered. Parallel to our findings, The ICAP believes that if the laboratory reported
such a pattern as negative, the additional information in the report on the pattern
and titer might go unnoticed because clinicians tend to pay less attention to
negative results [2].
We also found that reporting cytoplasmic reticular/linear patterns as ANA
positive led to a higher ENA reflex testing rate than ANA negative reports
(p-value<0.05). ENA reflex tests were recommended for nuclear ANA patterns,
which are more common than cytoplasmic patterns, as The ICAP recommends reporting
nuclear ANA patterns as positive [17].
We found that reporting cytoplasmic ANA patterns as positive was not enough for
clinicians to read the reflex test recommendations by The ICAP. Clinicians may pay
attention to the result and not the additional information in the report [2]. If a
clinician received a positive ANA report and didn’t pay attention to the
pattern/notes, he may assume it was a nuclear pattern, since nuclear
patterns are more frequent and always reported as positive, and would therefore
order the ENA reflex test. Paying attention to the results but not the
pattern/notes could be a reason for requesting the ENA reflex test. Else,
clinicians could read the results, pattern/titer information, and
recommendation notes, and still could order the ENA test according to the clinical
data of the patient. In our study, the positivity rate of ENA tests was too low
(1.16% and 0.00%) to support the second scenario.
ICAP declared that some rheumatologists thought that ANA patterns were not so
important, they just payed attention to the ANA results and the titer; second,
patterns were considered as irrelevant, because with an ANA positive, most of the
doctors would order anti-ENA tests; third, information obtained from staining
patterns was subjective, and varied according to the reader and dilution. This
thesis explained our study results. In our sttudy we found that the doctors who did
not pay attention to the patterns also did not pay attention to the notes.
Advice resulting from this study on reflex testing could be redundant until Turkey
develops a national reimbursement policy. Before solving the reimbursement problems,
advice about reflex testing could be redundant. After development of national
regulations, we could achieve efficient reflex test numbers by decreasing
unnecessary orders and increasing the appropriate requests. Unnecessary ENA reflex
tests conducted at the same visit as ANA testing could be reduced by this
regulation. The cost of non-effective ENA reflex tests could be decreased
(12.97% in the case of a positive ANA test and 1.10% in the case of
a negative ANA test). More importantly, the chance of probable diagnosis could be
increased, particularly in cases where clinicians do not read testing recommendation
notes (59.73% of positive ANA reports and 93.39% of negative ANA
reports). In a study, Among 108 laboratory respondents, 55 (51%) adopt the
practice of reflex testing (tests for specific autoantibodies, performed after a
positive ANA, and without a new order from the prescribing physician) or follow-up
testing (the same, after a new prescription). Some laboratories practicing the
reflex testing strategy integrate their ANA results into a general autoimmune report
or directly uploaded from the LIS into the patient’s EMR, with all specific
autoantibody tests conducted, technique used, and respective result. So a covetable
ratio of ideal reflex test numbers could be achieved. But this is not feasible in
many countries: the physician must order specific tests in a second step, according
to the ANA pattern reported and recomented reflex tests.
The ANA-reflex test is different from other reflex tests in terms of its algorithm
construction. Although it has very low predictive values, it serves a role in the
diagnosis of several systemic autoimmune rheumatic diseases as a first-line test
[17]. Additionally, the first test can be positive in up to 20–30%
of healthy subjects, especially some regular patterns as dense fine speckled-70,
which are not associated with systemic autoimmune disorders [17, 20, 21]. Despite
these drawbacks, the application of ANA-reflex testing has many objective
advantages, such as simplifying the patient workup, with only a single visit to the
doctor’s surgery and laboratory required, enabling a more rapid clinical
diagnosis. the ANA-reflex algorithm allows for more appropriate use of second-level
testing [3].
As an alternative to the national reimbursement policy for the reflex cascade
algorithm, the Italian model could be applied, which decreases bureaucratic or
administrative problems. Laboratories can calculate the cost of reflex tests by
considering the number and type of further tests. The fee for the ANA-reflex test
covers all of the possible second line tests, so additional payment problems can be
avoided [18]. An efficient workflow for reflex testing at the auto-immunology
laboratory must be compatible with the growing request for autoimmune diagnostic
tests, regional restrictions, and limited reimbursement [22]. We can adapt the
ANA-reflex request modality to our national health policy and culture to provide
rapid, complete diagnostic information with a critical impact on the clinical
decision [17].
Positive reporting of cytoplasmic reticular/linear ANA patterns resulted in
an incremental increase in AMA/ASMA test ordering (p-value<0.05),
and an incremental increase in ENA test ordering (p<0.05) into
consideration.
Reporting of cytoplasmic pattern with the ANA test has been suggested by some groups.
The Italian Forum Interdisciplinare per la Ricerca nelle Mallattie Autoimmuni
(FIRMA) and the second Brazilian consensus ICAP (although this changed in the third
consensus) recommended a cytoplasmic pattern to be considered ANA positive. However,
the European Consensus Finding Study Group on Laboratory Investigation in
Rheumatology (ECGSG), a member of the European League Against Rheumatism (EULAR),
advocates cytoplasmic pattern to be considered ANA negative due to the problems
during reimbursement, classification criteria, and EQC programs; simply put, there
is a conflict with the reporting of the test [2]. The major concern with respect to
reporting cytoplasmic patterns as ANA positive is that in some jurisdictions,
existing guidelines and diagnostic/classification criteria for systemic
lupus erythematosus (SLE), Sjogren’s syndrome, mixed connective tissue
disease (MCTD), systemic sclerosis (SSc), and autoimmune hepatitis (AIH) [13, 14]
are based on restricting ANA to nuclear patterns. Problematically, no guidance has
been provided about the interpretation of cytoplasmic and mitotic patterns.
Diagnosis can be affected by positive reported cytoplasmic and mitotic ANA patterns
[2, 4, 23–30]. Diagnostic criteria for AIH clearly define that only nuclear
patterns are to be interpreted as ANA [28–30]. A positive AMA result gives a
negative 4 points [28–30]. This scoring results in a paradox if AMA is
reported as ANA positive [2, 4, 28]. A third problem relates to the external quality
control (EQC) programs that accept cytoplasmic patterns reported as ANA negative.
With some alternative immunoassays, surprisingly, ACR and EASI/IUIS
recommendations allow a positive result with relevant cytoplasmic ANA patterns
[2].
At a multinational study, it was found that there were more expert-level laboratory
professionals (61%) than competent-level laboratory professionals
(46%) that considered cytoplasmic patterns as ANA positive (p<0,05).
The fraction of laboratory professionals that considered cytoplasmic ANA patterns as
ANA positive was higher in non-European countries (63%) than in European
countries (48%) (p<0,05). The reasons was maintained for historical
name problem (the name ‘antinuclear’ for the HEp-2 cell IIF test
does not take into consideration that autoantibodies to cell compartments other than
the nucleus) as well as for laboratory coding and invoicing [31, 32].
As written in an ICAP publication [50], jurisdictional and reimbursement reasons may
be at play in many countries, so that there is no universal consensus of the ICAP
experts for reporting cytoplasmic patterns.
Conclusion
Achieving an interim consensus between the clinical societies for
diagnostic/classification criteria in distinct diseases, as well as
political solutions to reimbursement policies, could decrease blockades to reporting
cytoplasmic and mitotic ANA patterns. In order to achieve this consensus between all
stakeholders, it is important to note that positive cytoplasmic ANA patterns have a
constructive influence on clinical perceptions but do not preclude the need to read
the recommendation notes. Not reading recommendation notes from positive cytoplasmic
ANA patterns could lead to various diagnostic and management problems.
