Frequency of Repeating Antinuclear Antibody Testing: When Less Is More

Objectives Antinuclear antibodies (ANA) are autoantibodies that are associated with and ordered to diagnose autoimmune connective tissue disease. ANA have high sensitivity (~98%) but low specificity (~75%), and because they can be found in healthy individuals and non-rheumatologic conditions leading to their elevation, ANA tests are often requested and interpreted inappropriately by clinicians. The aim of this study was to retrospectively assess how frequently ANA testing is repeated in the adult population of Saudi Arabia (SA) and which factors are associated with and lead to inappropriate testing. Methodology We investigated a study group of 40,634 adult patients who underwent 229,825 ANA tests from 2018 to 2022 in an academic hospital in Jeddah, SA. We took a random sample of 500 patients from the study group, along with their 998 ANA tests, to look in depth into our research questions. Variables related to patients, ANA tests, and ordering physicians were collected. Descriptive and analytical statistics were employed to address the research questions, and a p-value < 0.05 was considered statistically significant. Results We found 57% of the ordered ANA tests to have positive results, with the most common titers of mild positivity being 1:80 and 1:160. Most repeated ANA tests were ordered with an interval of more than one year, and when repeated, 67% of test results remained unchanged. The majority of seroconversions resulted from negative ANA tests or those with weak (titer 1:40) or mild positivity (titer 1:80-1:160). The results of the moderate (titer 1:320-1:640) and strong (titer ≥1280) positivity ANA tests did not change. Only 11% of repeated ANA tests were found to be appropriate for repetition. The most common specialties associated with ordering ANA tests in general were internal medicine, followed by rheumatology, and finally family medicine. Our correlation analysis revealed that being female, having systemic connective tissue disease, and having a rheumatologist as a specialist were all associated with ordering more than 10 ANA tests (p < 0.05). Conclusion Because the results of repeated ANA tests did not change much, our study suggests that the cost of repeating ANA tests and the subsequent potentially unnecessary interventions should all be carefully examined before scheduling a repeated ANA test. Further studies involving patients from SA and across wider healthcare settings (academic, community, and private hospitals and healthcare centers) are warranted.


Introduction
Laboratory investigations are a fundamental part of diagnosing, treating, and monitoring diseases in modern medicine [1].However, tests can be ordered inappropriately, resulting in either overutilization (ordering tests more than needed) or underutilization (omitting tests while clinically indicated) [2].Overutilization can also occur in two ways: tests are ordered at a patient's initial meeting or repeated after they have already been performed recently.A meta-analysis found that 20% of ordered tests were overutilized [2], while single studies (Cadamuro et al.) found that to be 70% [3].
Although laboratory investigations only represent 2.3% and 1.4% of total healthcare expenditure (HCE) in the United States and Germany, respectively [4], the downstream payment of laboratory investigations is undoubtedly expected to be larger, especially if inappropriately ordered, as overutilization can lead to unwanted sample-collection procedures, incorrect diagnoses, unnecessary additional investigations, and longer hospital stays.As such, multiple strategies and campaigns have been implemented to reduce unnecessary laboratory investigations [5].
Antinuclear antibodies (ANA) are a group of autoantibodies that are associated with and used to diagnose autoimmune connective tissue diseases, such as systemic lupus erythematosus (SLE), systemic sclerosis, primary Sjögren syndrome, mixed connective tissue disease, and idiopathic inflammatory myopathies (such as polymyositis and dermatomyositis) [6].ANA bind the nucleic acids DNA and RNA and their associated proteins and complexes.ANA and their autoantigens form immune complexes, which get implanted into tissues to damage cells and induce cytokine production and complement activation [7].
ANA are often tested as a group, and results are reported either positive or negative with a titer and immunofluorescence-staining pattern.Additional testing is then carried out to define the specific ANA present, which can be antibodies to Sm, double-stranded DNA (dsDNA), SSA/Ro60, U1RNP, topoisomerase I, centromere protein B (CENPB), RNA polymerase III, and Jo1 [6].
ANA at a titer of ≥1:80, for the first time, have been added in 2019 to the diagnosis criteria of SLE [8].The corresponding sensitivity and specificity of that titer are 97.8% and 74.7%, respectively [9]; ANA can be present in healthy individuals and certain populations, which can be as high as 50% [10], explaining the low specificity of such a low titer concentration.
Because testing for ANA is frequently performed unnecessarily by clinicians in patients presenting with symptoms such as widespread pain and fatigue, the American Society for Clinical Pathology and American Society for Clinical Laboratory Science recommended preserving the test for patients, with a high pre-test probability for connective tissue disease [11], as a positive result can be present in a healthy subject.Moreover, following positive results in patients with a connective tissue disease, rheumatologists often repeat ANA testing unnecessarily, and as a result, the American College of Rheumatology and British Society for Rheumatology also are against repeating ANA testing unless the disease picture has changed [12,13].
In this study, we retrospectively assess how frequently ANA testing is repeated in the adult population of Saudi Arabia (SA) and which factors are associated with and lead to inappropriate testing.

Study design, setting, and time
This was a retrospective study conducted at King Abdulaziz University Hospital (KAUH), Jeddah, Saudi Arabia (SA) from July 2023 to September 2023.

Study participants
The inclusion criteria were patients admitted to KAUH and who underwent ANA testing from 2018 to 2022.The exclusion criteria were patients who were aged less than 18 years or with incomplete data.

Data collection and sampling technique
ANA testing Current Procedural Terminology (CPT) codes (11521 for ANA profile and ANA pattern and 3854 for ANA antibodies) were used in the KAUH Laboratory database to retrospectively find patients who met our inclusion criteria.The KAUH Laboratory database retrieved 40,634 patients who had undergone 229,825 ANA tests from 2018 to 2022.Simple information regarding the demographics of each patient, the International Classification of Diseases (ICD)-coded diagnosis at the time of ordering the ANA tests, and specialties and locations of the ordering physicians of the ANA tests were also collected from the database.From the 40,634 patients (hereafter referred to as the study group), a random sample of 500 patients was selected to thoroughly search the electronic health records (EHRs) of each patient to collect the complete variables of interest for the study.Patient-related variables included demographic information about the patients, comorbidities, and the presence of connective tissue disease.ANA test-related variables included the number of ordered ANA tests, results, interval between tests, seroconversion of ANA tests (change of ANA results from positive to negative and vice versa), and order location.Variables associated with physicians included the corresponding specialties of the clinicians who ordered the ANA test for the patient.

Data handling
Nationality and residency variables were divided into "Saudi" vs. "non-Saudi" and "Jeddah" vs. "non-Jeddah," respectively.ICD diagnosis codes extracted from the KAUH Laboratory database were categorized into their main categories using WHO's ICD-10 online application (2019 version) [14].Connective tissue diseases were grouped according to their organ involvement into systemic diseases (such as SLE, rheumatoid arthritis (RA), and scleroderma), organ-specific (such as myasthenia gravis, gouty arthritis, and multiple sclerosis), and vasculitides.
ANA test results and titers were provided by the KAUH Laboratory and registered in the EHRs.In the random   1).The results of the χ2 test indicated a highly significant association between nationality and year (χ² = 459.825,df = 4, p < 0.001), suggesting a substantial variation in the distribution of nationalities across the study years, especially during the years impacted by the COVID-19 pandemic.

Diagnosis at the Time of Ordering
In examining the main diagnostic categories with a total percentage greater than 3%, several noteworthy trends emerge (  Factors influencing health status and contact with health services (total: 15.06%):The percentage in this category remained relatively consistent over the years, with a noticeable decrease in 2022 compared to previous years, which may reflect changes in health behavior after the COVID-19 pandemic.
Diseases of the musculoskeletal system and connective tissue (total: 12.61%): The percentage exhibited consistent fluctuations over the years, indicating potential trends that warrant further investigation.
Mental, behavioral, and neurodevelopmental disorders (total: 10.27%):The percentage remained relatively stable, with a slight decrease in 2022.
Diseases of the genitourinary system (total: 8.83%): Fluctuations in percentages, particularly in 2018 and 2022.
Symptoms, signs, and abnormal clinical and laboratory findings (total: 7.92%): The percentage increased over the years, peaking in 2022.Statistical analysis is essential to assess the significance of this trend.
Endocrine, nutritional, and metabolic diseases (total: 6.79%):The percentage increased over the years, with a noticeable rise in 2022.The last two categories have grown in the last few years.This could be related to the side effects of COVID-19 post-pandemic treatment.

Episode Type at the Time of Ordering
Among the participants who underwent ANA testing, the distribution demonstrated a predominant presence of outpatient episodes, followed by inpatient and emergency episodes (

Ordering Departments and Services of ANA Tests
The distribution of ANA tests across various departments provided insights into the healthcare landscape within the study (Table 4).The most prevalent department based on the provided data was internal medicine (Department 106), with a frequency of 112,799, which emerged as the most frequently utilized facility, constituting a substantial 49% of the total tests.Family medicine (Department 22) was also prominently featured, conducting 28,774 tests, accounting for 12.52% of the total.This department held a notable position in the distribution of ANA tests.Among the departments, the distribution of services revealed a diverse array of responses, with certain services standing out based on their count and percentage in the total dataset (Table A2

ANA Tests
The randomly selected sample of 496 patients underwent 998 tests over the years.Out of the patients, 317 (63.9%) had only one ANA test, and in total, 36.1% (179) patients had repeated ANA tests (Figure 1).ANA, antinuclear antibodies.
Positive results constituted 57% of the total 998 ANA test results (  ANA, antinuclear antibodies. Table 7 shows that, when positive, most of the titers were of mild positivity of 1:80 (22%) and 1:160 (13%).This pattern was similar to whether the patient did only one ANA test or whether his or her tests were repeated.We found that only 334 (33%) of the total ordered 998 ANA tests were negative (including those with a titer of 1:40), 98 (10%) were unreported, and 566 (57%) were positive (Table 6).The most common type of seroconversion was from positive results to negative ones, which happened to 9% (count 41 out of 479) of repeated tests.Mild-positivity (1:80-1:160) ANA tests constituted most of the change (Table 8).Of the 479 repeated ANA tests examined, only 25 (5%) ANA tests had their results changed from negative to positive.Our data revealed that ANA tests with moderate positivity (1:320-1:640) and strong positivity (≥1280) results tended not to seroconvert, as that happened to 1.7% and 0%, respectively, of reported tests.While looking at patients' whole pictures and correlating that with ANA test results and intervals, to assess whether repeating ANA tests more than once can be justified, we found that in 83% of repeated ANA tests (count 391 out of the 479 repeated tests), reordering ANA tests was unnecessary.In 6% of the assessed repetitions (count 31 repeated ANA tests), the data were not enough to judge this outcome.Only in 11% of the repeated ANA tests (count 50), the repetition could be justified (Figure 2A).Of the 50 instances in which we found repeating ANA tests was reasonable, 42% (count 21 repeated ANA tests) were due to laboratoryrelated issues, while 38% (count 19) were due to new symptoms that could be related to a yet-to-bediagnosed connective tissue disease.Only in 22% of the instances (count 11), repeating ANA tests led 11 patients to be diagnosed with connective tissue disease for the first time, as ANA tests were reordered (Figure 2B).

Correlational Analysis of Repeated ANA Tests
Our analysis found that patients with repeated ANA tests had a significantly higher percentage of being a Jeddah resident (p < 0.05) and having no connective tissue disease (72.1% vs. 27.9%)(Table 9).Of those patients who had ≥10 ANA tests, the majority of them were females and had connective tissue disease with systemic involvement (p < 0.05) (Table 10).Table 11 shows that rheumatology, out of all specialties, had a significantly higher percentage of physicians who ordered more than 10 ANA tests (p < 0.05).Lastly, we found that patients who had an organ-specific connective tissue disease had a significantly higher percentage of having ≥10 ANA tests or repeated ANA tests in general (p < 0.05) (

Discussion
ANA are autoantibodies that, when tested, have high sensitivity (~98%) but low specificity (~75%) against autoimmune diseases [9].The low specificity is likely due to ANA appearing in healthy individuals and getting elevated as a part of other non-rheumatologic conditions, such as malignancies, drug exposure, and infectious diseases [15].
When, in 2012, the American Board of Internal Medicine Foundation launched the campaign Choosing Wisely®: Next Steps in Improving Healthcare Value to encourage delivering evidence-based medicine among specialties and reduce cost and harm associated with unnecessary and unsupported use of investigations and treatments, ANA testing appeared at the top of the five items on which rheumatologists internationally agreed their practices needed to be questioned [16,17].Since then and all over the globe, multiple studies have been carried out to investigate the landscape of ANA testing: out of all ANA tests, how many are ordered appropriately; which specialties order most of the tests; how much unnecessary ANA tests represent of HCE; and what interventions can be used to reduce the inappropriate ordering of ANA tests [18][19][20].
One of the studies was done in Ontario, Canada, and involved 587,357 ANA tests that were performed on 437,966 adult patients from 2008 to 2015 [18].The researchers found that only 21.5% of ordered ANA tests were positive, and from 587,357 ANA tests, 28% (164,913 tests) were repeated, with almost half of the repeated (49%) reordered within a year from a previous test.Family medicine and rheumatology were the most common specialties to order ANA tests, representing 61% and 11%, respectively, of requesting specialties.Yeo et al. [19] in Australia went further and assessed the trends of 23,438 ANA tests that were performed on 19,603 patients from 2013 to 2020 and found that 95.7% of repeated tests were stable and their results did not change over time.
In this study, we retrospectively investigated similar research questions and tried to expose the landscape of ANA testing in an academic hospital in Jeddah, SA.Our study group consisted of 40,634 adult patients who underwent 229,825 ANA tests from 2018 to 2022.A random sample of 496 patients, who performed 998 ANA tests, was selected to answer the research questions in depth.We found that the majority of the patients were females (74.5% in the study group vs. 73.6% in the random sample), most patients were of SA nationality (70.1% in the study group vs. 81.9% in the random sample), and the mean age, in years, was 45.14 in the study group vs. 47.9 in the sample group.
We found the most common diagnoses associated with ordering ANA tests were "Factors influencing health status and contact with health services" (15.06%) (Table A1, Appendix, provides examples), followed by "Diseases of the musculoskeletal system and connective tissue" (12.61%)."Mental, behavioral, and neurodevelopmental disorders" represented 10.27%, "Diseases of the genitourinary system" represented 8.83%, and "Symptoms, signs, and abnormal clinical and laboratory findings" represented 7.92%."Endocrine, nutritional, and metabolic diseases" encompassed 6.79% of ANA testing-associated diagnoses.
Contrary to the above-mentioned studies [18,19], in our study, most ANA tests were often ordered by highly specialized specialists rather than family medicine doctors (in the study group, 49% of ordered ANA tests were by the internal medicine department vs. 12.52% for that by the family medicine department).The most common locations for ordering ANA tests were outpatient settings (67.7% of ordered ANA tests), followed by inpatient departments (21.4%), and emergencies came in last with 10.9% of the total ordered ANA tests.Similar trends appeared in our analysis of the random sample.This clearly reflects the current medical practice in the SA healthcare system, where there is room for improving organization and integration of the system, to avoid patients accessing highly specialized care prior to primary care evaluation [21].
Within the sample group, we found 57% of ANA tests (count 566 out of 998 tests) to be positive, with the most common titer results of mild positivity of 1:80 (22%) and 1:160 (13%).The percentage was similar whether the patient had only one ANA test or the tests were repeated.These findings are in disagreement with the previously mentioned studies, as the majority of ANA tests were found to be negative rather than positive [18,19].Such findings likely reflect the variability in the cut-off values used to define a positive test, which needs further assessment.
Our analysis revealed that after repetition, most of the results of repeated tests (67%) do not change, and if there was a change, the majority was from positive test results to negative ones.Most seroconversion came from negative ANA tests or those with weak (titer 1:40) or mild positivity (titer 1:80-1:160).Moderate (titer 1:320-1:640) and strong (titer ≥1280) positivity ANA tests tended to not change.These patterns of seroconversion can be explained by the ups and downs of ANA in non-rheumatologic diseases, which are more common than rheumatologic diseases [15]; and our results reinforce the notion that ANA tests do not need to be repeated unless the disease picture has changed [11][12][13].When examining whether repetition of ANA tests can be justified, we found that in 83% of the instances, repeating the test was not appropriate.Only in 11% of instances (count 50), repeating ANA tests was suitable, and in 42% (count 21 out of 50), that was related to laboratory or sampling issues.Of the 179 patients who got their ANA tests repeated, repeating ANA tests led 11 patients to be diagnosed with connective tissue diseases for the first time.
Lastly, we performed a correlation analysis and found that being a Jeddah resident and having no connective tissue disease were associated with a higher percentage of getting repeated ANA tests in general.On the other hand, being a female, having a systemic connective tissue disease, and the specialist being a rheumatologist were all associated with ordering more than 10 ANA tests.
Our study has limitations.First, the studied patients and examined ANA tests were retrieved from a single academic and specialized hospital, which may have affected the numbers and results of performed ANA tests, as sicker patients are often the most served patients, which contrasts community and private practices.Second, some variables related to patients, such as comorbidities, socioeconomic status, death, and diagnosis at the time of the ANA test order, were not incorporated in-depth or at all in our analysis.Third, and although small, some ANA tests have missing data, such as the results and intervals.Lastly, we only included the specialties of the ordering physicians, but many variables (such as the clinician's demographics, level of education, graduation program/school, and whether the repeated and initial ANA tests were ordered by the same physician or not) were not assessed.Further studies involving patients from SA and across diverse healthcare settings are needed before conclusions are drawn.

Conclusions
The study's aim was to assess the landscape of ANA tests and the factors that lead to the inappropriate repetition of ANA tests.Some of our findings were in alignment with other studies in the literature, while others were not.But the conclusion was the same: in most cases, ANA results mostly do not change, and repeating the tests is futile.ENT: ear, nose, and throat surgery; OBS: obstetrics; GYNE: gynecology.

Appendices
* In King Abdulaziz University Hospital (KAUH), most rheumatologists have duties in the Clinical Teaching Units (CTUs), which are part of the Medical Services of the Internal Medicine Department.As such, the KAUH database registered them as "Medical Services" doctors and not "rheumatologists," hence their representation is small.While collecting the data for the sample group of 500 patients, we were able to separate them.In the sample group, rheumatologists ordered more ANA tests than family medicine doctors.

FIGURE 1 :
FIGURE 1: Percentage distribution of the repeated ANA tests and their numbers (groups) (N = 496).

FIGURE 2 :
FIGURE 2: Distribution of the justification outcome of repeated ANA tests (N = 479).

(
A) Was there a need for ANA tests to be repeated?(B) If repeating an ANA test was reasonable, what was the justification?ANA, antinuclear antibodies; CTD, connective tissue disease.

Table 2 )
(Table A1in the Appendix provides the most common diagnoses in each category).

Table 3 )
. Emergency episodes (E) were observed in 25,250 instances, representing 10.9% of the total dataset.Inpatient episodes (I) accounted for 49,696 occurrences, making up 21.4% of the total dataset.The majority of episodes fell under the outpatient category (O), with 156,872 instances comprising 67.7% of the total dataset.

selected sample of 500 patients and 998 ANA tests
7% of the total dataset.Ophthalmology service (Service 48) was notable, with a count of 5,355, representing 2.3% of the total responses.Similarly, the orthopedic surgery service (Service 49) accounted for 3.2% of the total responses, with a count of 7,512.Urology service (Service 51) was also significant, contributing 3,523 responses and representing 1.5% of the total.
, Appendix).Noteworthy services, each accounting for over 1% of the total responses, are as follows: medical services (Service 106, including rheumatology) of the internal medicine department emerged prominently with a count of 42,184, constituting a significant 18.2% of the total responses.Within the broader category of the internal medicine department, nephrology (Service 106.14) held a substantial presence, accounting for 14,431 instances, or 6.2% of the total.Hematology service (Service 109) was a significant contributor, with a count of 15,963, representing 6.9% of the total.General arrhythmia service (Service 140) was noteworthy, contributing 6,610 responses and constituting 2.9% of the total.Ob/Gyn service (Service 107) was notable, comprising 19,783 responses and contributing to 8.5% of the total dataset.Surgical service (Service 108) represented 5.2% of the total responses, with a count of 11,963.Emergency unit services (Service 129) stood out with a count of 18,335, making up 7.9% of the total responses.General clinics service of the family medicine department (Service 22) played a prominent role, with 17,848 instances, making up 7.comorbidities (diabetes, hypertension, cardiovascular disease, etc.) and 14.7% had connective tissue diseases; of them, 61.7% had a disease with a systemic nature (SLE, RA, scleroderma, and Sjögren-Larsson syndrome), while 34.2% had organ-specific diseases (myasthenia gravis, bilateral flexor tenosynovitis, gouty arthritis, multiple sclerosis, myopathy, osteoarthritis, psoriasis, sacroiliitis, and fibromyalgia) and 4.1% had vasculitides (Behcet disease).Of patients who had SLE (total: 23), seven (30.4%) had lupus nephritis (Table5).Variable No. (%) Age (years) (mean ± SD) 47.9 ± 15.53If the answer is "Yes," specify: (N = 73)

TABLE 5 : Distribution of studied participants according to their demographic characteristics, comorbidity, connective tissue diseases, and SLE, as well as lupus nephritis comorbidity (N = 496).
SLE, systemic lupus erythematosus.

Table 6
).When repeated (count 479 ANA tests), the interval between tests was mostly more than one year, but results remained unchanged in 67% of repeated tests (count 323 tests out of 479 repeated tests).The most common specialties to request ANA testing were internal medicine and rheumatology, representing 50% (546 ANA tests) and 21% (215 tests), respectively, of ordering specialties.Family medicine represented 8% (82 tests) of ordered ANA tests.The majority of the ANA tests were ordered from the outpatient (O) settings (78%), followed by inpatient (14%) and emergency (E) (8%) departments (Table6).VariableNo.(%

TABLE 13 : The most common diagnoses that appeared in our database from the ICD-10 online application (2019 version) of the WHO (N = 229,825).
ICD, International Classification of Diseases; WHO, World Health Organization.