Abstract
Background:
Current American College of Rheumatology/European League Against Rheumatism (ACR-EULAR) guidelines indicate a positive biopsy with a score of ≥1 using focus scores (FS) to quantify lymphocytic foci presence. Accurate labial minor salivary gland biopsy (LMSGB) diagnosis depends on proper biopsy sampling, observer interpretation, and FS. There are fewer studies that evaluate the sensitivity and specificity of histology and serological testing. There was a substantial association between FS and the serological test, according to one study, but not in another.
Aim:
To determine the diagnostic sensitivity and specificity of LMSGB following the current ACR/EULAR diagnostic recommendations used for patients with suspected Sjögren’s disease (SjD).
Materials and Methods:
Patients who had the symptoms of either dry mouth/eyes or both or suspected SjD with extraglandular symptoms and underwent minor salivary gland biopsies were included in this study. The study analyzed the variables of adequate amount of biopsy samples, focal scores, interpretations among pathologists with demographical data, and biochemical analysis for SjD diagnosis.
Results:
The positive predictive and negative predictive values were low, indicating limited effectiveness in predicting biopsy results. The overall accuracy was 40.54%, indicating ineffectiveness in distinguishing between those with and without the condition.
Conclusion:
The present study highlights the diagnostic limitations of minor salivary gland biopsy, indicating that a negative result does not rule out SjD but can rule out other infiltrative causes of xerostomia.
Keywords: Accuracy, focal score, interobserver variability and autoimmune
Introduction
Sjögren’s disease (SjD) is a systemic chronic inflammation, characterized by lymphocytic infiltration in exocrine organs, joints, gastrointestinal tract, central nervous system, and other organs. The immune system’s interactions with lymphocyte infiltration and plasma cell activation cause glandular hyposecretion, resulting in symptoms such as generalized parotid gland enlargement, xerostomia, and xerophthalmia, with autoantibodies and soluble inflammatory mediators. SjD is divided into primary and secondary categories, with primary being an isolated sicca disease without underlying autoimmune disease and secondary being associated with other autoimmune disease. The diagnosis is challenging due to the overlapping clinical presentations.
The Current American College of Rheumatology/European League against Rheumatism (ACR-EULAR) guidelines indicate a positive biopsy with a score of ≥1 using focus scores (FS) to quantify lymphocytic foci presence.[1] Accurate labial minor salivary gland biopsy (LMSGB) diagnosis depends on proper biopsy sampling, observer interpretation, and FS.[2] Sjögren’s International Collaborative Clinical Alliance criteria require positive serum antibodies, an ocular staining score of 3 or greater, and lymphocytic sialadenitis on a minor salivary gland biopsy. Histological features of SjD from LMSGB are seen as a key component in determining a definitive diagnosis. Interpretation of the minor salivary gland biopsy in the diagnosis of SjD can alter the sensitivity and predictive value of the histopathological criteria.[3,4] The purpose of the current study is to evaluate the validity of LMSGB reporting in accordance with the most recent ACR/EULAR diagnostic guidelines for individuals who may have Sjögren’s syndrome.
Materials and Methods
This cross-sectional study carried out in the Department of Oral and Maxillofacial Pathology at Sri Ramachandra Dental College and Hospital between January 2018 and 2023 to address the research question. The study population consisted of patients referred to establish a diagnosis of SjD. Other medical conditions such as head-and-neck radiation, hepatitis C infection, human immunodeficiency viral infection, sarcoidosis, amyloidosis, and graft-vs-host disease were excluded. This study was approved by the institutional ethics committee (IEC-NI/23/AUG/88/53). Histopathology reports and slides of LMSGB along with demographics and clinical and laboratory findings were collected by two investigators in a standardized manner [Figure 1]. The pathologists reviewed the literature on salivary gland biopsy interpretation to establish a systematic approach. The following parameters used to diagnosis SS be evaluated micromorphologically with salivary gland sample that met the glandular tissue adequacy criterion. The evaluation of LMSGB microscopical features for the presence of salivary gland lobules number, focal lymphocytic sialadenitis was performed. One focus is an aggregate of >50 lymphocytes present in the periductal area and the focal score is the total number of foci per 4 mm2 [Figure 2]. The histological pattern of one or more foci in the biopsies, with the surrounding tissue primarily constituted of unaffected parenchyma, is known as focal lymphocytic sialadenitis. The study involved collecting H and E-stained slides which were blinded and graded independently by two pathologists, with disagreements resolved by group re-evaluation. The amount of glandular tissue and glandular surface area was calculated using low-power and high-power magnification. The number of foci observed in each 4 mm2 area was scored as 0, 1, 2, and 3.
Figure 1.
Diagnostic criteria flowchart of Sjögren’s disease
Figure 2.
Focal score in Hematoxylin and Eosin stain
Data collection and analysis
An Excel spreadsheet was used to record the original LMSGB histopathological slides with the revised interpretations to examine the data from the biopsy interpretation. Statistical analysis was performed using IBM SPSS software version 21.0 (IBM, Armonk, NY, USA). Descriptive data were expressed in mean ± standard deviation median (min–max). The categorical data were compared using the Pearson Chi-square test. P < 0.05 was considered statistically significant.
Results
One hundred and forty-eight patients included in the present study of the LMSGB met the inclusion criteria. The mean age was 44.5 years (range 17–79 years). One hundred and twenty-five patients were women (85%) while 15% were males [Table 1]. Dry mouth and eyes were the most common chief complaint of the study population. About 48% of patients were not associated with autoimmune illness while 51% with a documented medical history of autoimmune disease involvement were among the 148 patients. About 1% of the biopsy sample was not adequate for histopathological evaluation [Table 2]. The standard histopathological observations were tabulated from two observers and analyzed statistically using kappa from level agreement; Kappa statistics was used to evaluate the level of agreement between two observers. When the interobservation statistics were determined, it was found to be 0.646, thereby indicating a substantial level of agreement between the two observers with a very minimal interobserver bias [Table 3].
Table 1.
Demographic, clinical and laboratory data
| Parameters | Biopsy report positive (n) | Biopsy report negative (n) |
|---|---|---|
| Age | 44.48±13.398 | |
| Gender | ||
| Male | 22 | |
| Female | 126 | |
| Clinical manifestation | ||
| With extra glandular involvement | 44 | 29 |
| With glandular involvement | 31 | 44 |
| Serum antibodies | ||
| Positive | 32 | 25 |
| Negative | 41 | 48 |
| No investigation performed | 2 | |
| Anti-rheumatoid factor | ||
| Positive | 26 | 30 |
| Negative | 36 | 41 |
| No investigation performed | 13 | 2 |
| CRP | ||
| Positive | 10 | 27 |
| Negative | 54 | 44 |
| No investigation performed | 11 | 2 |
| Anti-CCP | ||
| Positive | 0 | 2 |
| Negative | 64 | 69 |
| No investigation performed | 11 | 2 |
| Ocular staining | ||
| Positive | 0 | 1 |
| Negative | 64 | 70 |
| No investigation performed | 11 | 2 |
| Tissue acquisition | ||
| Inadequate | 0 | 2 |
| Adequate | 146 | 0 |
CRP: C-reactive protein; CCP: Cyclic citrullinated peptide
Table 2.
Impact of tissue acquisition for analysis on labial minor salivary gland biopsy outcomes
| Acquisition of tissue for analysis | Histopathological analysis - 4–6 lobules |
Total | |
|---|---|---|---|
| Absent | Present | ||
| Inadequate | 2 (1.35) | 0 | 2 (1.35) |
| Adequate | 0 | 146 (98.6) | 146 (98.6) |
| Total | 2 (1.35) | 146 (98.6) | 148 (100.0) |
Table 3.
Inter-rater reliability assessment using kappa statistic
| Scoring | Observer 2 |
Total, n (%) | Kappa | P | |||
|---|---|---|---|---|---|---|---|
| 0.00, n (%) | 1.00, n (%) | 2.00, n (%) | 3.00, n (%) | ||||
| Observer 1 | |||||||
| 0.00 | 74 (97.4) | 2 (2.6) | 0 | 0 | 76 (100.0) | 0.646 | <0.001 |
| 1.00 | 11 (28.2) | 21 (53.8) | 7 (17.9) | 0 | 39 (100.0) | ||
| 2.00 | 2 (7.7) | 7 (26.9) | 17 (65.4) | 0 | 26 (100.0) | ||
| 3.00 | 0 | 1 (14.3) | 2 (28.6) | 4 (57.1) | 7 (100.0) | ||
| Total | 87 (58.8) | 31 (20.9) | 26 (17.6) | 4 (2.7) | 148 (100.0) | ||
The diagnostic performance of a glandular and extraglandular manifestation and definitive biopsy results across 148 records. The evaluation correctly identified 41.33% of cases positive in glandular manifestation but had a high rate in extraglandular symptoms. Its specificity was 39.726%, with low specificity and high false positives in extraglandular symptoms. The positive likelihood ratio was 0.6858, indicating limited disease confirmation but a role in ruling out diseases. The positive predictive and negative predictive values were low, indicating limited effectiveness in predicting biopsy results. The overall accuracy was 40.54%, indicating ineffectiveness in distinguishing between those with and without the condition [Table 4].
Table 4.
Diagnostic performance of biopsy results in clinical suspected Sjögren’s disease
| Parameter | Value (%) |
|---|---|
| Sensitivity | 41.33 |
| Specificity | 39.726 |
| Positive likelihood ratio | 0.6858 |
| Negative likelihood ratio | 1.4768 |
| PPV | 41.33 |
| NPV | 39.73 |
| Accuracy | 40.54 |
PPV: Positive predictive value; NPV: Negative predictive value
All the cases with no investigation (100%) resulted in a positive biopsy report. Among those who tested negative for the specific serum antibodies, 48 cases (53.9%) had a negative biopsy report, while 41 cases (46.1%) had a positive biopsy report. Among those who tested positive for the specific serum antibodies, 25 cases (43.9%) had a negative biopsy report and 32 cases (56.1%) had a positive biopsy report. The data suggest that there is not a statistically significant association between the serum antibodies presence and biopsy report outcomes, given the P = 0.184.
Among noninvestigated cases, 2 cases (13.3%) had a negative biopsy report, while a significant majority, 13 cases (86.7%), resulted in a positive biopsy report. Around 41 cases with negative rheumatoid factor (RF) test, 53.2% had a negative biopsy report and 36 cases with negative RF test (46.8%) had a positive biopsy report. For individuals with positive RF test results, 30 cases (53.6%) had a negative biopsy report and 26 cases (46.4%) had a positive biopsy report. The P = 0.013 indicates the statistical significance of the association between RF test results and biopsy report outcomes.
Among the study samples, noninvestigated cases – 2 (15.4%) had a negative biopsy report, while the majority, 11 cases (84.6%), resulted in a positive biopsy report. In the negative C-reactive protein (CRP) test group, 44 cases (44.9%) had a negative biopsy report and 54 cases (55.1%) had a positive biopsy report. For individuals with positive CRP test results, 27 cases (73.0%) had a negative biopsy report and 10 cases (27.0%) had a positive biopsy report. The P value (0.0001) indicates the statistical significance of the association between CRP levels and biopsy report outcomes.
Among cases where anti-cyclic citrullinated peptide (Anti-CCP) antibody test was not conducted, 2 cases (15.4%) had a negative biopsy report, while the majority, 11 cases (84.6%), resulted in a positive biopsy report. Among individuals with negative Anti-CCP antibody test results, 69 cases (51.9%) had a negative biopsy report and 64 cases (48.1%) had a positive biopsy report. For individuals with positive Anti-CCP antibody test results, both cases (100.0%) had a negative biopsy report, indicating a 100% proportion of negative biopsy outcomes in this group. The P value (0.015) indicates the statistical significance of the association between anti-CCP antibody test results and biopsy report outcomes.
Among cases where no ocular staining score was conducted, 2 cases (15.4%) had a negative biopsy report, while the majority, 11 cases (84.6%), resulted in a positive biopsy report. Among individuals with negative ocular staining scores, 70 cases (52.2%) had a negative biopsy report and 64 cases (47.8%) had a positive biopsy report, indicating a slightly higher proportion of negative biopsy outcomes among individuals with negative scores. For individuals with positive ocular staining scores, 1 case (100.0%) had a negative biopsy report, indicating a 100% proportion of negative biopsy outcomes in this group. The P value (0.024) indicates the statistical significance of the association between ocular staining score and biopsy report outcomes.
Discussion
To date, LMSGB represents the gold standard for the diagnosis of SjD. Despite being an invasive process, it is also used to diagnose the disorders associated to IgG4, sarcoidosis, and amyloidosis. FS is a semiquantitative tool used to assess inflammatory infiltration of the salivary gland parenchyma’s tissue per square millimeter [Figure 2]. FS has a preponderant role in both ACR/EULAR diagnostic criteria.[1] The diagnostic accuracy of a histopathological report is largely dependent on the quality of tissue samples and the interpretation of the histological findings.[5] Fisher et al. and Fox recommend submitting a minimum 8 mm2 glandular surface area for interpretation to minimize the sampling error and ensure that 4–6 lobules are submitted for lymphocytic foci location.[6,7] In the present study, consistent pathology results were found that 146 out of 148 available glandular tissue samples, with 99% positive and 1% negative LMSGBs. Histopathological features such as acinar atrophy, ductal dilation, fibrosis, and adipocyte replacement are observed in patients over 50 years, as well as in young patients with high focal scores. These fibrosis caused by tissue damage, triggers immune responses, leading to inflammatory reactions and the epithelial-mesenchymal transition, transforming epithelial cells into mesenchymal-like cells, contributing to chronic autoimmune disorders such as Sjögren disease.[8] The report’s main weakness is its reliance on expert opinion, but a balance of accuracy can be achieved with two observers.[6,9,10] The number of glandular lymphocytic foci in these studies had an inter-observer agreement value (kappa score) of 0.646. These results are consistent with results from previous study.[11] Shiboski et al.’s study found that 7% of patients with SjD had a change in FS from negative to positive, 11% had a change from positive to negative, and 82% had no change[12] The sensitivity and specificity of LMSGB are variable, but a meta-analysis found a high diagnostic value for primary SjD, with a specificity of 88% and sensitivity of 78%.[13] Gioveli et al. in their study found that revaluation findings demonstrate the system’s excellent diagnostic accuracy and statistical significance in predicting biopsy report outcomes.[14] In contrast, the present study, the sensitivity and specificity found to be 41.33% and 39.72%, respectively. This low value attributed to small size of the population.
The diagnosis approaches of this multifacet disease include clinical, laboratory, imaging, and pathological investigations, including tests for RF, anti-citrullinated protein antibody, and anti-nuclear antibodies.[14] The diagnostic performance of a clinical evaluation compared to definitive biopsy results. The results of the definitive biopsy showed a substantial discrepancy from clinical assessment, according to this study. 60.3% of cases extraglandular manifestation had positive biopsy findings. Positive clinical glandular manifestation was made from 58.7% of negative biopsy results. The present study yielded 41.33% positive predictive value and 38.72% negative predictive value indicating that clinical judgment may not always align with definitive biopsy findings. This may be the result of varying clinical patterns, extraglandular involvement, and varying degrees of inflammation and cytokine production on two distinct hands. Although autoantibodies are present in two-thirds of suspected SS patients, LMSGB is typically recommended for establishing a diagnosis of SS in the absence of autoantibodies.[15] This study found that 46.1% of cases tested negative for specific serum antibodies had a positive biopsy report, indicating no significant association between serum antibody presence and biopsy report outcomes. The ocular staining score and biopsy report outcomes were found to be statistically significant, as evidenced by the P = 0.024. Our findings agreed with those of Atkinson et al.[16] The study found a significant association between RF and CRP test results and biopsy report outcomes, with 53.2% and 44.9% cases having negative biopsy reports and 46.8% and 55.1% having positive reports, with a P = 0.013 and 0.001, respectively. With a P = 0.015, the current study’s Anti-CCP antibody test results and biopsy report outcomes are statistically significant. The RF, anti-CCP, and CRP positivity could be due to comorbidities that helps to classify the primary and secondary SS.[17]
Limitation
The primary constraints of the current investigation are its single-center setup, retrospective methodology, and lack of molecular biology assay.
Conclusion
The LMSGB is a crucial diagnostic tool for SjD, aiding in differential diagnosis of sarcoidosis and amyloidosis. However, its diagnostic accuracy for SjD is low, limiting clinical applications. Future research involving larger patient populations is required to accurately assess the diagnostic value of both LMSGB and recently developed new diagnostic tools. The positive likelihood ratio is 0.6858, indicating limited disease confirmation.
Conflicts of interest
There are no conflicts of interest.
Acknowledgments
The authors wish to acknowledge Dr. Ramaprabha for her statistical service of this study.
Funding Statement
Self-funding.
References
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