Cited by 26Systemic Lupus Erythematosus ResearchBlood Coagulation and Thrombosis MechanismsClinical Laboratory Practices and Quality Control

Lupus anticoagulant mixing tests for multiple reagents are more sensitive if interpreted with a mixing test‐specific cut‐off than index of circulating anticoagulant

Open access
Kumano, Osamu, Moore, Gary W.·2018·Research and Practice in Thrombosis and Haemostasis

Abstract

Unlabelled Box •Various approaches are available for interpreting lupus anticoagulant mixing tests. •Mixing test specific cut‐off (MTC) was compared with index of circulating anticoagulant (ICA). •MTC was superior to ICA in detecting inhibition by lupus anticoagulant (LA) in multiple reagents. •Detection rates of LA can be improved by the method to interpret the results. Background Lupus anticoagulant (LA) is classified in the antibody family that is recognized as antiphospholipid antibodies. Guidelines for LA detection recommend mixing test interpretation with either a mixing test specific cut‐off (MTC) or index of circulating anticoagulant (ICA). We previously evidenced that MTC was superior to ICA in detecting the in vitro inhibition of LA with a single dilute APTT (activated partial thromboplastin time) and dRVVT (diluted Russell's viper venom time) pairing. Objectives The objective in the present study was to compare the LA diagnostic effectiveness of MTC and ICA by multiple APTT and dRVVT reagents. Methods One hundred‐five samples from non‐anticoagulated patients positive for LA in the dilute APTT (dAPTT) and dRVVT reagent pairing employed for diagnostic examination were performed by undiluted and in a 1:1 mix with normal pooled plasma with four additional APTT reagents and another dRVVT reagent (dRVVT B). Results Frequencies of MTC and ICA positivity were determined from samples LA positive in undiluted plasma. MTC positivity in mixing test were 63%, 77%, 80%, 84%, 46%, 81%, and 72% in 4 APTT, dAPTT and 2 dRVVT, respectively. ICA positivity were 47%, 67%, 58%, 54%, 42%, 47%, and 29%, respectively. There were no samples of ICA‐positive/MTC‐negative with any reagent. Conclusions The data indicate that MTC is superior to ICA for LA detection in mixing tests in multiple reagents and reagent types. Although mixing tests may make weak LA samples appear negative, the efficacy of LA detection can be improved by the method to interpret the results.

AI snapshot

Written from the paper’s full text.

TL;DR

In 105 non-anticoagulated patients already positive for lupus anticoagulant (LA), interpreting 1:1 mixing tests with a mixing test-specific cut-off (MTC) detected more LA than the index of circulating anticoagulant (ICA) across multiple APTT and dRVVT reagents. No sample was ICA-positive/MTC-negative, supporting MTC as the more sensitive mixing-test interpretation.

Key findings

  • MTC positivity in mixing tests was 63%, 77%, 80%, 84%, 46%, 81%, and 72% across the four APTT reagents, dAPTT, and two dRVVT reagents, respectively; ICA positivity was 47%, 67%, 58%, 54%, 42%, 47%, and 29%, respectively.
  • There were no samples that were ICA-positive/MTC-negative with any reagent.
  • In undiluted plasma, LA positivity ranged from 33% to 63% for APTT-based assays and 62% to 70% for dRVVT assays.
  • Mix ratio and ICA values were significantly higher in samples positive by both MTC and ICA than in MTC-only or both-negative groups in all reagents (P < .01).

Methods

  • Plasma samples from 105 non-anticoagulated patients who were LA-positive on routine diagnostic testing: 44 with definite APS, 12 with SLE and persistent LA, 9 with SLE and LA-positive at testing, and 40 undergoing diagnostic testing for aPL/APS.
  • Samples were tested undiluted and in 1:1 mixes with normal pooled plasma using four additional APTT reagents and another dRVVT reagent (dRVVT B), alongside the routine dAPTT and dRVVT reagents.
  • Cut-offs were derived from 50 normal samples; comparisons used Wilcoxon signed rank tests with P < .01 considered significant, and reference intervals were calculated as mean ± 2 SD.

Limitations

  • The paper states that mixing tests were assessed in the “ideal” situation of otherwise uncompromised samples, and that further work is planned in other situations including anticoagulant therapy and factor deficiencies, with ROC curve analysis for cut-off generation.
  • The paper notes that mixing tests introduce a dilution factor and may make weak LA samples appear negative.
  • Evident from the text: because only LA-positive samples were studied, the findings address sensitivity of MTC versus ICA but do not establish specificity or performance in LA-negative or unselected populations.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1002/rth2.12069

Cites

42 of 42 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

10 of 31 cited sources checked · 7 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    Mixing tests may make weak LA samples appear negative due to a dilution factor.

    “mixing plasma with NPP shows a dilution factor that can make weaker LA samples appear negative in the mixing test despite clear positivity in the screening and confirmation results on undiluted plasma”
    • Also stated in: § Conclusions · hedged
      “Although mixing tests may make weak LA samples appear negative”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [7] Keeling D, Mackie I, Moore GW, Greer IA, Greaves M; British Committee for Standards in Haematology. Guidelines on the investigation and management of antiphospholipid syndrome. Br J Haematol. 2012; 157: 47– 58. 22313321 source
    • cites [8] Ledford‐Kraemer MR, Moore GW, Bottenus R, Danniele C, de Groot PG, Exner T. Anticoagulant Approved Guideline. 1st ed CLSI Document H60. Wayne, PA: Clinical and Laboratory Standards Institute; 2014.

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [13] Devreese KM. Interpretation of normal plasma mixing studies in the laboratory diagnosis of lupus anticoagulants. Thromb Res. 2007; 119: 369– 76. 16704874 source
  • Premisecitation

    In situations where alternative or co-existing coagulation abnormalities are present, mixing tests can improve specificity of LA testing.

    “However, in situations where alternative or co‐existing coagulation abnormalities are present, mixing tests can improve specificity of LA testing.34, 35, 36, 37”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [34] Devreese KMJ, Laat BD. Mixing studies in lupus anticoagulant testing are required at least in some type of samples. J Thromb Haemost. 2015; 13: 1– 4. 26017873 source
    • cites [35] Martinuzzo ME, Barrera LH, Dadamo MA, Otaso JC, Gimenez MI, Oyhamburu J. Frequent false‐positive results of lupus anticoagulant tests in plasmas of patients receiving the new oral anticoagulants and enoxaparin. Int J Lab Hematol. 2014; 36: 144– 50. 24034808 source
    • cites [36] Pennings MT, de Groot PG, Meijers JC, Huisman A, Derksen RH, Urbanus RT. Optimisation of lupus anticoagulant tests: should test samples always be mixed with normal plasma? Thromb Haemost. 2014; 112: 736– 42. 25008787 source
  • Premisecitation

    Potency alone does not necessarily correlate with clinical significance and thrombotic risk.

    “On the other hand, a number of studies have indicated populations of clinically significant LA despite the negative results in the mixing tests14, 32, 36 and the potency alone does not necessarily correlate with clinical significance and thrombotic risk.37, 39”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [37] Devreese KMJ. No more mixing tests required for integrated assay systems in the laboratory diagnosis of lupus anticoagulant? J Thromb Haemost. 2010; 8: 1120– 2. 20149080 source
    • cites [39] Devreese K, Peerlinck K, Hoylaerts MF. Thrombotic risk assessment in the antiphospholipid syndrome requires more than the quantification of lupus anticoagulants. Blood. 2010; 115: 870– 8. 19965629 source
  • Findingown data

    Positivity by both MTC and ICA was higher with FSL than all other reagents in the study.

    “In addition, positivity by both MTC and ICA was higher with FSL than all other reagents in the study.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The ICA cut-off values established with the routinely employed LA reagents are applicable to the additional APTT and dRVVT reagents.
  • Findingown data

    Mix ratio values of MTC only positive samples were significantly higher than those negative in both MTC and ICA in all reagents.

    “In addition, the values of MTC only positive were significantly higher than those of negative in both MTC and ICA in all reagents.”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 10
  • May combine two claims. 4
  • The quote only partly states this claim. 1
  • Classified as probably not a substantial claim.

    Premisecitation

    Guidelines recommend that mixing tests are interpreted with either MTC or ICA.

    “Guidelines recommend mixing tests are interpreted with either a mixing test specific cut‐off (MTC) or the index of circulating anticoagulant (ICA).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [6] Pengo V, Tripodi A, Reber G, et al. Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis. Update of the guidelines for lupus anticoagulant detection. J Thromb Haemost. 2009; 7: 1737– 40. 19624461 source
    • cites [7] Keeling D, Mackie I, Moore GW, Greer IA, Greaves M; British Committee for Standards in Haematology. Guidelines on the investigation and management of antiphospholipid syndrome. Br J Haematol. 2012; 157: 47– 58. 22313321 source

      The Lupus anticoagulant testing section says mixing tests may be used to detect an inhibitor, and the Recommendation section describes interpreting an abnormal screening result from a mixture. Neither passage recommends interpreting mixing tests with an MTC or ICA; the paper’s discussion of cut-offs concerns LA tests generally, not those two methods specifically. Page numbers are unavailable in our copy. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [8] Ledford‐Kraemer MR, Moore GW, Bottenus R, Danniele C, de Groot PG, Exner T. Anticoagulant Approved Guideline. 1st ed CLSI Document H60. Wayne, PA: Clinical and Laboratory Standards Institute; 2014.

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    There is no single test to detect all LA, and guidelines recommend performance of two different tests (dRVVT and LA-sensitive APTT).

    “There is no single test to detect all LA1, 6, 7, 8 and guidelines recommend performance of two different tests that represent different assay principles, the diluted Russell's viper venom time (dRVVT) and a LA‐sensitive activated partial thromboplastin time (APTT).6, 7, 8”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [1] Meroni PL, Borghi O, Raschi E, Tedesco F. Pathogenesis of antiphospholipid syndrome: understanding the antibodies. Nat Rev Rheumatol. 2011; 7: 330– 9. 21556027 source
    • cites [6] Pengo V, Tripodi A, Reber G, et al. Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis. Update of the guidelines for lupus anticoagulant detection. J Thromb Haemost. 2009; 7: 1737– 40. 19624461 source
    • cites [7] Keeling D, Mackie I, Moore GW, Greer IA, Greaves M; British Committee for Standards in Haematology. Guidelines on the investigation and management of antiphospholipid syndrome. Br J Haematol. 2012; 157: 47– 58. 22313321 source
  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Integrated testing is sufficient to detect LA without performing the mixing test in samples with no other causes of clotting time prolongation and LA confirmatory test results within reference intervals.

    “For samples from patients with no other causes of clotting time prolongation and LA assay confirmatory test results within reference intervals, the increasingly popular paradigm of integrated testing is sufficient to detect the presence of LA without performing the mixing test at all.6, 7, 8, 9, 26, 27, 28, 31, 33, 34”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [6] Pengo V, Tripodi A, Reber G, et al. Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis. Update of the guidelines for lupus anticoagulant detection. J Thromb Haemost. 2009; 7: 1737– 40. 19624461 source

      The Integrated tests section says these tests combine screening and confirmation and, in principle, do not require a mixing test. It does not establish that they are sufficient specifically when other causes of prolonged clotting time are absent and confirmatory results fall within reference intervals. Its statement is also more qualified than the citing sentence’s assertion of sufficiency. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites [7] Keeling D, Mackie I, Moore GW, Greer IA, Greaves M; British Committee for Standards in Haematology. Guidelines on the investigation and management of antiphospholipid syndrome. Br J Haematol. 2012; 157: 47– 58. 22313321 source

      The Lupus anticoagulant testing section says that, when no other cause of prolonged clotting time is present, samples may be considered LA-positive based on positive screening and confirmatory tests on undiluted plasma, despite a negative mixing test. It does not specify that the confirmatory test result must fall within its reference interval, and it recommends confirmation with a fresh sample whenever possible. Page numbers are unavailable in our copy. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [8] Ledford‐Kraemer MR, Moore GW, Bottenus R, Danniele C, de Groot PG, Exner T. Anticoagulant Approved Guideline. 1st ed CLSI Document H60. Wayne, PA: Clinical and Laboratory Standards Institute; 2014.

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Findingown data

    The percentage of LA detected in the FSL screening test was 57%, higher than SP (38%) and SLA (33%), and similar to Cephen and PTT silica-based reagents.

    “In the present study, performed on a much larger cohort of LA‐containing plasmas, the percentage of LA detected in the FSL screening test was 57%, much higher than the 38% and 33%, respectively, of SP and SLA, and similar to that of Cephen and PTT silica‐based reagents.”
    Quote in the paperSubstantial claim
  • May combine two claims.

    Findingcitation

    Screen and mix ratios and ICA values were similar between the two dRVVT reagents.

    “Screen and mix ratios and ICA values were similar between the two dRVVT reagents, possibly due, at least in part, to normalizing the data reducing between reagent differences.28, 29”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [28] McGlasson DL, Fritsma GA. Comparison of six dilute Russell viper venom time lupus anticoagulant screen/confirm assay kits. Semin Thromb Hemost. 2013; 39: 315– 9. 23468380 source
    • cites [29] Jennings I, Greaves M, Mackie IJ, Kitchen S, Woods TA, Preston FE; UK National External Quality Assessment Scheme for Blood Coagulation. Lupus anticoagulant testing: improvements in performance in a UK NEQAS proficiency testing exercise after dissemination of national guidelines on laboratory methods. Br J Haematol. 2002; 119: 364– 9. 12406070 source
  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    LA2 confirmation reagent confirmed LA activity in undiluted plasma for all samples with elevated LA1 screening results.

    “Similarly, LA2 confirmation reagent (Siemens Healthineers) confirmed LA activity in undiluted plasma for all samples with elevated LA1 screening results.”
    Quote in the paperNot a substantial claim
  • The quote only partly states this claim.

    Interpretationown data

    Detection rates of LA can be improved by using MTC rather than ICA to interpret mixing test results.

    “Detection rates of LA can be improved by the method to interpret the results.”
    • Also stated in: § Conclusions · plain
      “the efficacy of LA detection can be improved by the method to interpret the results.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The ICA cut-off values established with the routinely employed LA reagents are applicable to the additional APTT and dRVVT reagents.
  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    All samples with elevated APTT screen ratios achieved significant correction by the Actin FS ratio to confirm LA activity in undiluted plasma.

    “All samples with elevated APTT screen ratios achieved significant correction by the Actin FS ratio to confirm LA activity in undiluted plasma.”
    Quote in the paperNot a substantial claim
  • Classified as probably not a substantial claim.

    Study design and conductcitation

    Cut-off values for screen, confirm, and mixing test in each additional reagent were determined from upper limits of the distribution of 50 normal samples in each reagent.

    “Cut‐off values for screen, confirm, and mixing test in each additional reagent were determined from upper limits of the distribution of 50 normal samples in each reagent, and for ICA with the routinely employed LA reagents, ICA cut‐off values were previously established and used in each additional reagent.9, 21, 22, 23”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [9] Moore GW, Culhane AP, Daw CR, Noronha CP, Kumano O. Mixing test specific cut‐off is more sensitive at detecting lupus anticoagulants than index of circulating anticoagulant. Thromb Res. 2016; 139: 98– 101. 26916303 source
    • cites [21] Kumano O, Ieko M, Naito S, Yoshida M, Takahashi N, Aoki T. Index of circulating anticoagulant cut‐off value establishment in activated partial thromboplastin time mixing test for lupus anticoagulant diagnosis. J Thromb Haemost. 2013; 11: 1919– 22. 23819748 source
    • cites [22] Kumano O, Ieko M, Naito S, et al. Verification of the guidelines for lupus anticoagulant detection: usefulness of index for circulating anticoagulant in APTT mixing test. Thromb Res. 2014; 134: 503– 9. 24935674 source
  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Factor deficiencies and undisclosed anticoagulation were excluded by performing coagulation screening tests prior to the LA assays.

    “Factor deficiencies and undisclosed anticoagulation were excluded by performing coagulation screening tests prior to the LA assays.”
    Quote in the paperSubstantial claim
  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    53.3% of the cohort had established persistent LA.

    “We performed the LA assays in a clinically select and appropriate cohort, 53.3% of whom had established persistent LA.”
    Quote in the paperNot a substantial claim
  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The population distributions for the normal donor samples were confirmed as Gaussian.

    “The population distributions for the normal donor samples were confirmed as Gaussian.”
    Quote in the paperNot a substantial claim
  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    The study specifically assessed mixing tests in the 'ideal' situation of otherwise uncompromised samples.

    “Thus, it is valuable to maximise diagnostic efficacy of mixing tests, so for the present study, we specifically assessed mixing tests in the “ideal” situation of otherwise uncompromised samples.”
    Quote in the paperSubstantial claim
  • May combine two claims.

    Interpretationreasoning

    Whilst integrated testing can be diagnostically accurate and logistically attractive, there are situations where additionally undertaking mixing tests achieves accurate and more confident diagnoses.

    “Whilst integrated testing can be diagnostically accurate and logistically attractive, there are situations where additionally undertaking mixing tests achieves accurate and more confident diagnoses.”
    Quote in the paperSubstantial claim
14 of 14
  • The paper relies on this without support.

    The ICA cut-off values established with the routinely employed LA reagents are applicable to the additional APTT and dRVVT reagents.

    “for ICA with the routinely employed LA reagents, ICA cut‐off values were previously established and used in each additional reagent.9, 21, 22, 23”
1 of 1

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • ICA has a high specificity to factor deficiency, whereas the mixing test ratio has a high sensitivity to inhibitors.

    “For instance, ICA has a high specificity to factor deficiency, whereas the mixing test ratio has a high sensitivity to inhibitors [6,7], although both indexes are recommended for LA diagnosis [8,9].”

    From Performance characteristics of 5 numerical indexes in mixing test interpretation under coexistence of lupus anticoagulant and coagulation factor deficiency (2023) · cites this paper as [6]

    The Discussion reports that a mixing test-specific cut-off (MTC) was more sensitive than ICA for detecting lupus anticoagulant, supporting the sensitivity comparison for this inhibitor. The Materials and Methods section says factor deficiencies were excluded before testing; the paper therefore does not establish that ICA has high specificity to factor deficiency. (LLM, conf=0.88)

    Experimental — accuracy not yet measured
  • Previous studies have improved the sensitivity and specificity by establishing suitable cutoff values.

    “Previous studies have improved the sensitivity and specificity by establishing suitable cutoff values [1,6].”

    From Performance characteristics of 5 numerical indexes in mixing test interpretation under coexistence of lupus anticoagulant and coagulation factor deficiency (2023) · cites this paper as [6]

    The Discussion (page unavailable) reports that a mixing-test-specific cut-off improved sensitivity compared with the index of circulating anticoagulant. It says mixing tests can improve specificity in certain circumstances, but does not show that establishing the cut-off improved specificity. (LLM, conf=0.85)

    Experimental — accuracy not yet measured
  • The mixing test normalized ratio is most sensitive to inhibitors.

    “The mixing test normalized ratio showed non-correction in all LA samples, indicating that this index is most sensitive to inhibitors”

    From Performance characteristics of 5 numerical indexes in mixing test interpretation under coexistence of lupus anticoagulant and coagulation factor deficiency (2023) · cites this paper as [6]

    The Discussion (page not provided) says that the mixing ratio interpreted with a mixing-test-specific cut-off was more sensitive than the index of circulating anticoagulant for detecting lupus anticoagulant inhibition. It does not establish that the ratio is most sensitive to inhibitors generally; the Results (page not provided) also show that not all lupus-anticoagulant-positive samples were positive in mixing tests. (LLM, conf=0.83)

    Experimental — accuracy not yet measured
  • Although MTC provides greater sensitivity for the detection of LA, especially when weak LA is present, it has a greater tendency to produce false-positive results than ICA.

    “Although MTC provides greater sensitivity for the detection of LA, especially when weak LA is present, it has a greater tendency to produce false-positive results than ICA [16,18,19].”

    From Comparison of different algorithms for lupus anticoagulant detection: a single-center experience (2024) · cites this paper as [18]

    The Results and Discussion report higher MTC sensitivity than ICA for detecting LA, including cases that mixing-test dilution could otherwise cause to appear negative. But the reported comparison uses samples positive for LA in undiluted plasma; it does not establish that MTC produces more false-positive results than ICA. No page numbers are available in our copy. (LLM, conf=0.88)

    Experimental — accuracy not yet measured
  • Performance characteristics in each index have been well examined in samples with a single cause of APTT prolongation, but those with 2 causes remain largely unknown.

    “Although performance characteristics in each index have been well examined in samples with a single cause of APTT prolongation [1,[5], [6], [7]], those with 2 causes (eg, factor VIII [FVIII] deficiency and LA) remain largely unknown.”

Cites

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Authors
Kumano, O., & Moore, G. W.
Affiliations
  • Sysmex (Japan) (JP)
  • St Thomas' Hospital (GB)
Corresponding author
Kumano, Osamu
Venue
Research and Practice in Thrombosis and Haemostasis
Publisher
Elsevier BV
Published
2018-01
Volume
2
Issue
1
Pages
105-113
ISSN
2475-0379 (ISSN-L)
OpenAlex
W2779707539
Cited by
26
References
44
Citations by year
2026: 1 · 2025: 4 · 2024: 2 · 2023: 6 · 2022: 4 · 2021: 2 · 2019: 5 · 2018: 1 · 2017: 1
FWCI
1.47
Type
journal article
Language
en
Open access
Locations
  • Research and Practice in Thrombosis and Haemostasis (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (submittedVersion, cc-by-nc-nd)
  • PubMed Central (submittedVersion, cc-by-nc-nd)
Topics
  • Health Sciences › Medicine › Rheumatology › Systemic Lupus Erythematosus Research
  • Health Sciences › Medicine › Hematology › Blood Coagulation and Thrombosis Mechanisms
  • Health Sciences › Medicine › Physiology › Clinical Laboratory Practices and Quality Control
Keywords
lupus anticoagulant, activated partial thromboplastin time, antiphospholipid antibodies

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