Patterns of Antiphospholipid Antibody Testing and Positivity in a Real-World Laboratory over Two Decades: The Role of IgM

Antiphospholipid antibody (aPL) testing is fundamental for diagnosing and classifying antiphospholipid syndrome (APS) under the Sydney criteria, which recommend the detection of lupus anticoagulant (LA), anticardiolipin antibody (aCL), and anti-β2-glycoprotein I (ab2GPI) antibodies in both IgG and IgM isotypes.[1] However, the clinical significance of IgM has remained uncertain. Comparative studies consistently show that IgG isotypes have stronger and more reproducible associations with thrombotic or obstetric events than IgM.[2] Reflecting this, the International Society on Thrombosis and Haemostasis (ISTH) Scientific and Standardization Committee (SSC) emphasized that while IgM testing is formally included, its interpretation is often complex, particularly in the absence of IgG or LA positivity, due to assay variability and limited outcome correlations.[3] This evolving evidence was incorporated into the 2023 American College of Rheumatology and the European Alliance of Associations for Rheumatology (ACR/EULAR) classification criteria, where isolated IgM positivity is assigned minimal laboratory weight due to its reduced specificity and frequent low-risk or false-positive profiles. Importantly, this weighting applies to classification only and does not alter the recommended diagnostic workup.[4]

While some reports find little correlation between isolated IgM and morbidity,[5] others note possible phenotype-specific links, such as obstetric events, retinal vein thrombosis, or cerebrovascular events in younger patients.[6] [7] [8] Large-scale work, such as Egiziano et al's analysis of over 33,000 patients, demonstrates the value of big-data approaches in uncovering real-world testing patterns, showing that many IgM-positive results were not reproducible on repeat testing and underscoring the limited reliability of isolated IgM positivity.[9] Our study expands this evidence by describing real-world aPL ordering patterns and testing results of LA, aCL, and anti-β2-glycoprotein, both IgG and IgM.

We reviewed all aPL results across Mayo Clinic sites in the United States from January 2006 to July 2025, aligning with the release of the revised Sapporo (Sydney) criteria. This was a laboratory-based analysis without incorporation of clinical events, and positive aPL laboratories should not be interpreted as a diagnosis of antiphospholipid syndrome. For each patient, we identified the earliest occasion on which any aPL test was performed. We extracted available results for LA, aCL IgG and IgM, and β2-glycoprotein I (b2GPI) IgG and IgM. Positivity was defined according to international criteria and internal standards: ≥40 units for aCL and b2GPI, and for LA, ISTH SSC guideline-based demonstration of a phospholipid-dependent inhibitor in Dilute Russell's viper venom time (DRVVT) or activated partial thromboplastin time (aPTT)-based platelet neutralization procedure, Staclot LA delta, and HEX LA delta assays, with specific cutoffs applied for each. Given the laboratory-based nature of this study, potential false positives could not be excluded. Positivity rates were calculated for individual markers, and a subset of patients who underwent complete testing for all three categories at initial workup were analyzed for single-, double-, and triple-positivity distributions, defined as positivity for one, two, or all three aPL categories (LA, aCL, or b2GPI, in either IgG or IgM isotype), respectively. We also identified patients positivity only for IgM isotypes (aCL or b2GPI) in the absence of IgG or LA.

We identified 99,814 patients tested for at least one aPL marker, with LA being the most frequently ordered test (n = 75,085; positivity rate = 10.3%), followed by aCL IgG (n = 69,539; positivity rate = 1.2%), aCL IgM (n = 69,640; positivity rate = 1.7%), b2GPI IgG (n = 37,791; positivity rate =1.9%), and b2GPI IgM (n = 37,601; positivity rate = 2.0%; [Table 1]). LA was the predominant laboratory investigation, while ordering of IgM isotypes was less frequent. The ordering patterns and overlaps between the five markers are shown in [Fig. 1].

Table 1 Positivity rates and distribution of antiphospholipid antibody test results at first-encounter testing, and impact of omitting IgM, IgG, or LA assays

Metric (first-encounter testing)	n/N	Percentage of cohort[a]
All patients tested (any aPL marker)	99,814	–
• Lupus anticoagulant positive	7,730/75,085	10.3
• aCL IgG-positive	846/69,539	1.2
• aCL IgM-positive	1,198/69,640	1.7
• b2GPI IgG-positive	732/37,791	1.9
• b2GPI IgM-positive	762/37,601	2.0
Fully tested subgroup[b]	30,274	–
• Triple-positive (LA + aCL + b2GPI)	407	1.3
• Double-positive	427	1.4
• Single-positive	3,009	9.9
• Isolated IgM-positive[c]	668	2.2
aCL IgM only	296	0.9
b2GPI IgM only	224	0.7
Both IgM only	148	0.5
Effect of omitting IgM tests
• Laboratory-positive patients missed[d]	668/3,843	17.4
• “Triple-positive” reclassified	140/407	34.4
• “Double-positive” reclassified to “single-positive”	132/427	30.9
• “Double-positive” reclassified to “negative”	148/427	34.7
Effect of omitting IgG tests
• Laboratory-positive patients missed[d]	307/3,843	8.0
• “Triple-positive” reclassified	228/407	56.0
• “Double-positive” reclassified to “single-positive”	112/427	26.2
• “Double-positive” reclassified to “negative”	21/427	4.9
Effect of omitting LA
• Laboratory-positive patients missed[d]	2,187/3,843	56.9
• “Triple-positive” reclassified	407/407	100.0
• “Double-positive” reclassified to “single-positive”	242/427	56.7
• “Double-positive” reclassified to “negative”	0/427	0.0

Abbreviations: aCL, anticardiolipin; aPL, antiphospholipid antibody; b2GPI, β2-glycoprotein I; LA, lupus anticoagulant.

^a Percentages use the relevant denominator in the “n/N” column.

^b Patients with simultaneous LA, aCL IgG/IgM, and b2GPI IgG/IgM results.

^c Positive for aCL IgM and/or b2GPI IgM with negative LA and IgG isotypes.

^d Number and proportion of all initially aPL-positive patients (single, double, or triple) who would not have been detected.

Fig. 1 Upset plot showing the overlap in test ordering for five aPL markers (LA, aCL IgG, aCL IgM, b2GPI IgG, and b2GPI IgM) in patients tested at the earliest occasion from 2006 onwards. aCL, anticardiolipin antibody; aPL, antiphospholipid antibody; b2GPI, β2-glycoprotein I; LA, lupus anticoagulant.

Among the 30,274 patients who were fully tested for all three marker categories (LA, aCL, and b2GPI), 1.3% (n = 407) were triple-positive, 1.4% (n = 427) were double-positive, and 9.9% (n = 3,009) were single-positive; the remaining 87.3% (n = 26,431) were negative for all markers ([Table 1]). Within the single-positive group, most patients had isolated LA (73.1%), while 9.9% (n = 296) had aCL IgM only and 7.5% (n = 224) had b2GPI IgM only ([Fig. 2]). Omitting IgM from the panels would have resulted in missing 17% (668/3,843) of patients with at least one positive aPL marker and in the reclassification of 34% (140/407) of those initially classified as triple positive, reflecting classification effects. These findings apply to initial laboratory profiles only and cannot be interpreted as missed APS diagnoses, as repeat testing and clinical data were not available for this study.

Fig. 2 Distribution of single-positive patients (n = 3,009). Most had isolated lupus anticoagulant positivity (73.1%), followed by aCL IgM (9.9%), b2GPI IgM (7.5%), b2GPI IgG (7.5%), and aCL IgG (2.1%). aCL, anticardiolipin antibody; aPL, antiphospholipid antibody; b2GPI, β-glycoprotein I; LA, lupus anticoagulant.

In this real-world analysis of the largest to date laboratory cohort of patients undergoing initial aPL testing over a 20-year timeframe, we found that LA was the most frequently ordered assay, followed by aCL IgG, aCL IgM, b2GPI IgG, and b2GPI IgM. This pattern aligns with previous studies in which LA was also the most frequently tested.[10] In contrast, Egiziano et al found that aCL was the most frequently ordered test, followed by LA and b2GPI, with only 19% of patients receiving simultaneous testing for all three criteria antibodies.[9] These comparisons suggest considerable variability in aPL ordering practices across regions, driven by institutional protocols and health care system factors. In our institution, LA may have resulted as part of the protocol for prolonged aPTT, without a clinical suspicion of APS, and additionally, its higher frequency is explained by the fact that LA is included in the thrombophilia panel, whereas aCL and b2GPI antibodies are not. Moreover, these ordering patterns diverge from the ISTH SSC recommendations, which advise performing LA, aCL, and ab2GPI assays together to aid interpretation.[1] The predominance of LA testing and the frequent use of incomplete panels in our cohort highlight a substantial gap between recommended practice and real-world testing behavior.

With respect to IgM antibodies, although only a small proportion of fully tested patients in our cohort had IgM-only positivity, their representation among all aPL-positive patients was higher, indicating that omission of IgM testing might substantially reduce the overall number of patients who could ultimately meet classification criteria for APS if they undergo repeat testing at 12 weeks and fulfill the required clinical features. Previous work has shown that IgM-only positivity is relatively uncommon in thrombotic APS but more frequent in obstetric APS, with isolated IgM rates of 3.5% to 5.4% in thrombotic cases and 5.7% to 12.3% in obstetric cases, depending on the assay used.[8] Moreover, specialized APS cohorts have reported that approximately 14% of triple-positive patients include IgM cases, meaning they would not be identified as such if IgM assays were excluded.[11] In our dataset, the proportion of triple-positive patients lost without IgM testing was notably higher, suggesting a potentially greater contribution of IgM to classification in broader, real-world populations. This study is limited by its cross-sectional nature for the evaluation of only initial laboratory testing. Positive aPL laboratories in this analysis do not equate to a diagnosis of APS. The importance of these other clinical factors and the need for follow-up aPL testing cannot be understated when it comes to understanding future thrombotic risk and adhering to guideline recommendations; despite these limitations, initial antithrombotic therapy decisions are often made when initial results are available, and therefore, the findings of even this cross-sectional analysis have important upfront clinical implications. Future work will focus on evaluating rates of repeat testing and their correlation with thrombotic outcomes.

This large-scale analysis of real-world aPL testing revealed that exclusive IgM positivity, defined as aCL or ab2GPI IgM in the absence of IgG and LA, represents a sizable group of patients with initial positive aPL testing, comparable with isolated IgG positivity. Further study is needed to understand how these results may impact APS diagnosis, which requires clinical criteria to be met and persistent antibody positivity after 12 weeks. As clinical criteria and repeat testing were not available in this dataset, these findings should be interpreted within the context of laboratory prevalence rather than APS diagnosis. These findings offer a comprehensive characterization of aPL positivity patterns in routine clinical practice and may support future refinements in diagnostic strategies.

Publication History

Received: 27 September 2025

Accepted: 23 December 2025

Article published online:
20 January 2026

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