CoChat
Sign Up
Cited by 4Hemophilia Treatment and ResearchBlood Coagulation and Thrombosis MechanismsSystemic Lupus Erythematosus Research

Performance characteristics of 5 numerical indexes in mixing test interpretation under coexistence of lupus anticoagulant and coagulation factor deficiency

Open access
Matsuda, Masato, Hoshiyama, Yoshiki, Ogawa, Kazuei, Emmi, Mari, Terai, Shuji, Moriyama, Masato·2023·Research and Practice in Thrombosis and Haemostasis
DOI 10.1016/j.rpth.2023.100065PMC PMC9986643
Open this paper in CoChat — free accountOpen PDF

Abstract

Background: The mixing test is useful to investigate the cause of unexpectedly prolonged activated partial thromboplastin time (APTT). Several indexes are available for distinguishing correction from non-correction (ie, factor deficiency from inhibitors), but their performance characteristics may differ because of their different formulas. Furthermore, it is unclear how each index performs under the coexistence of factor deficiency and inhibitors. Objectives: The objective of this study was to examine the differences in indexes, depending on factor VIII activity (FVIII:C) levels and lupus anticoagulant (LA) titers in test samples. Methods: APTT was measured in spiked samples with various FVIII:C levels and LA titers, normal pooled plasma (NPP), and their 4:1, 1:1, and 1:4 mixtures. The following 5 indexes were calculated: index of circulating anticoagulant, mixing test normalized ratio, 4:1 and 1:1 percent corrections, and an APTT difference between the 1:1 mixture and NPP. The samples with LA, showing correction, were measured for FVIII:C in a one-stage assay to check parallelism. Results: All indexes showed correction under FVIII deficiency and non-correction under higher LA titers. However, under lower LA titers, some indexes showed non-correction but others showed correction because of dilution effects and variations in formulas and/or sample mix ratios. The differences among the indexes were more pronounced under coexistent FVIII deficiency and LA, even though LA titers were equal in the tested samples; samples with lower FVIII:C showed correction, whereas those with normal FVIII:C showed non-correction. The samples tested for FVIII:C showed non-parallelism. Conclusion: Each index had different performance characteristics to LA samples, which were pronounced under low FVIII:C levels in test samples.

AI snapshot

Written from the paper’s full text.

TL;DR

This in vitro study compared five numerical indexes for interpreting APTT mixing tests when lupus anticoagulant (LA) and factor VIII deficiency coexist. It found that index performance diverged especially at lower FVIII:C levels, with some indexes calling correction while others called non-correction despite equal LA titers.

Key findings

  • All five indexes showed correction in FVIII-deficient samples without anti-PS/PT, and non-correction at higher LA titers.
  • In LA samples, the mixing test normalized ratio showed non-correction in all samples, whereas ICA and Mix–NPP showed correction at lower LA titers; 1:1 and 4:1 percent correction showed non-correction at lower LA titers under normal FVIII:C.
  • Under coexisting FVIII deficiency and LA, lower FVIII:C samples were more often judged as correction, while normal FVIII:C samples were non-correction, even when LA titers were equal.
  • One-stage FVIII assays showed non-parallelism in anti-PS/PT-containing samples, including those with lower FVIII:C.

Methods

  • Spiked plasma samples were prepared from normal pooled plasma and FVIII-deficient plasma, with anti-PS/PT monoclonal IgG added to create 20 samples combining 4 anti-PS/PT concentrations (0, 25, 37.5, and 50 μg/mL) and 5 FVIII:C levels (92.1, 46.2, 22.8, 11.8, and 6.1 IU/dL); LA titers by dRVVT screen/confirm ratio were 1.10, 1.47, 1.58, and 1.75.
  • APTT mixing tests without incubation were performed in duplicate using a CP3000 analyzer and Thrombocheck APTT-SLA reagent at test plasma:NPP ratios of 1:1, 4:1, and 1:4.
  • Five indexes were calculated—ICA (cutoff 12.4), mixing test normalized ratio (cutoff 1.12), 1:1 percent correction (cutoff 72), 4:1 percent correction (cutoff 50), and Mix–NPP (cutoff 8)—and one-stage FVIII assays with dilutions from 1/5 to 1/80 were used to check parallelism.

Limitations

  • The paper states that only spiked plasma samples were tested, only one combination of reagents and analyzer was used, and cutoff values may depend on that combination; results should be validated with clinical samples.
  • Evident from the text: the findings are descriptive comparisons of in vitro spiked samples, with no clinical outcome data or formal statistical testing reported.

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

Integrity signals

Registry

Crossref · 10.1016/j.rpth.2023.100065

Cites

23 of 23 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

13 of 28 cited sources checked · 9 supported · 0 flagged · accuracy not yet measured

  • Interpretationown data

    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”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [6] Kumano, 2017, Lupus anticoagulant mixing tests for multiple reagents are more sensitive if interpreted with a mixing test-specific cut-off than index of circulating anticoagulant, Res Pract Thromb Haemost source

      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
    • cites [7] Kumano, 2019, Ruling out lupus anticoagulants with mixing test-specific cutoff assessment and the index of circulating anticoagulant, Res Pract Thromb Haemost source

      The Introduction reports that the mixing test–specific cutoff (based on the mix ratio) was more sensitive to lupus anticoagulants than the index of circulating anticoagulant. The Results also found more frequent inhibitor detection with that cutoff for most, but not all, reagents. This paper does not report non-correction in all lupus anticoagulant samples or establish that the ratio is the most sensitive index overall. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [8] Ledford-Kraemer, 2014, Anticoagulant Approved Guideline

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

  • Interpretationcitation

    No indexes permit 100% accurate discrimination.

    “because no indexes permit 100% accurate discrimination [3].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [3] Favaloro, 2020, Coagulation mixing studies: utility, algorithmic strategies and limitations for lupus anticoagulant testing or follow up of abnormal coagulation tests, Am J Hematol source
    • Rests on an assumption: The cutoff values used for the five indexes are appropriate for the specific APTT reagent and analyzer combination used in this study.
  • Interpretationcitation

    Coagulation factor deficiency strongly influences the results of mixing tests.

    “Our results also suggest that coagulation factor deficiency strongly influences the results of mixing tests [10].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [10] Devreese, 2015, Mixing studies in lupus anticoagulant testing are required at least in some type of samples, J Thromb Haemost source
    • Rests on an assumption: The cutoff values used for the five indexes are appropriate for the specific APTT reagent and analyzer combination used in this study.
  • Findingown data

    All five indexes showed correction in samples with lower FVIII:C levels without anti-PS/PT.

    “In samples with lower FVIII:C levels without anti-PS/PT, all indexes showed correction (Figure 1)”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The cutoff values used for the five indexes are appropriate for the specific APTT reagent and analyzer combination used in this study.
  • Findingown data

    All five indexes showed non-correction under higher LA titers.

    “All indexes showed correction under FVIII deficiency and non-correction under higher LA titers.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The cutoff values used for the five indexes are appropriate for the specific APTT reagent and analyzer combination used in this study.
  • May combine two claims. 10
  • Classified as probably not a substantial claim. 10
  • The quote only partly states this claim. 2

  • May combine two claims.

    Premisecitation

    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.”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [1] Kershaw, 2013, Mixing tests: diagnostic aides in the investigation of prolonged prothrombin times and activated partial thromboplastin times, Semin Thromb Hemost source
    • cites [5] Chang, 2002, A “percent correction” formula for evaluation of mixing studies, Am J Clin Pathol source
    • cites [6] Kumano, 2017, Lupus anticoagulant mixing tests for multiple reagents are more sensitive if interpreted with a mixing test-specific cut-off than index of circulating anticoagulant, Res Pract Thromb Haemost source

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

    Premisecitation

    Correction generally indicates coagulation factor deficiency, whereas non-correction suggests the presence of an inhibitor.

    “Correction generally indicates coagulation factor deficiency, whereas non-correction suggests the presence of an inhibitor (eg, lupus anticoagulant [LA] or a specific coagulation factor inhibitor) [3].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [3] Favaloro, 2020, Coagulation mixing studies: utility, algorithmic strategies and limitations for lupus anticoagulant testing or follow up of abnormal coagulation tests, Am J Hematol source

  • May combine two claims.

    Premisecitation

    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].”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [6] Kumano, 2017, Lupus anticoagulant mixing tests for multiple reagents are more sensitive if interpreted with a mixing test-specific cut-off than index of circulating anticoagulant, Res Pract Thromb Haemost source

      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
    • cites [7] Kumano, 2019, Ruling out lupus anticoagulants with mixing test-specific cutoff assessment and the index of circulating anticoagulant, Res Pract Thromb Haemost source

  • Classified as probably not a substantial claim.

    Premisecitation

    The mixing test is useful to investigate the cause of unexpectedly prolonged activated partial thromboplastin time (APTT).

    “The mixing test is useful to investigate the cause of unexpectedly prolonged activated partial thromboplastin time (APTT).”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [1] Kershaw, 2013, Mixing tests: diagnostic aides in the investigation of prolonged prothrombin times and activated partial thromboplastin times, Semin Thromb Hemost source
    • cites [2] Kershaw, 2017, Performance and interpretation of mixing tests in coagulation, Methods Mol Biol source
    • cites [3] Favaloro, 2020, Coagulation mixing studies: utility, algorithmic strategies and limitations for lupus anticoagulant testing or follow up of abnormal coagulation tests, Am J Hematol source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    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].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [1] Kershaw, 2013, Mixing tests: diagnostic aides in the investigation of prolonged prothrombin times and activated partial thromboplastin times, Semin Thromb Hemost source
    • cites [6] Kumano, 2017, Lupus anticoagulant mixing tests for multiple reagents are more sensitive if interpreted with a mixing test-specific cut-off than index of circulating anticoagulant, Res Pract Thromb Haemost source

      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

  • Classified as probably not a substantial claim.

    Premisecitation

    Numerical indexes are reportedly useful in observed correction.

    “Because the correction/non-correction interpretation is subjective, numerical indexes are reportedly useful in observed correction [[1], [2], [3], [4], [5]].”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [1] Kershaw, 2013, Mixing tests: diagnostic aides in the investigation of prolonged prothrombin times and activated partial thromboplastin times, Semin Thromb Hemost source
    • cites [2] Kershaw, 2017, Performance and interpretation of mixing tests in coagulation, Methods Mol Biol source
    • cites [3] Favaloro, 2020, Coagulation mixing studies: utility, algorithmic strategies and limitations for lupus anticoagulant testing or follow up of abnormal coagulation tests, Am J Hematol source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    LA is identified in patients with antiphospholipid syndrome and also other diseases such as coronavirus disease 2019.

    “LA is identified in patients with antiphospholipid syndrome [14,24] and also other diseases such as coronavirus disease 2019 [25], in which anti-PS/PT is detected [26].”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [14] Atsumi, 2000, Association of autoantibodies against the phosphatidylserine-prothrombin complex with manifestations of the antiphospholipid syndrome and with the presence of lupus anticoagulant, Arthritis Rheum source
    • cites [24] Meroni, 2011, Pathogenesis of antiphospholipid syndrome: understanding the antibodies, Nat Rev Rheumatol source
    • cites [25] Le Joncour, 2021, Antiphospholipid antibodies and thrombotic events in COVID-19 patients hospitalized in medicine ward, Autoimmun Rev source

  • May combine two claims.

    Findingown data

    The differences among indexes were more pronounced under coexistent FVIII deficiency and LA, even though LA titers were equal in the tested samples.

    “The differences among the indexes were more pronounced under coexistent FVIII deficiency and LA, even though LA titers were equal in the tested samples”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The cutoff values used for the five indexes are appropriate for the specific APTT reagent and analyzer combination used in this study.

  • May combine two claims.

    Findingown data

    Under lower LA titers, some indexes showed non-correction but others showed correction.

    “However, under lower LA titers, some indexes showed non-correction but others showed correction because of dilution effects and variations in formulas and/or sample mix ratios.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The cutoff values used for the five indexes are appropriate for the specific APTT reagent and analyzer combination used in this study.

  • May combine two claims. The quote only partly states this claim.

    Findingown data

    Samples with lower FVIII:C showed correction, whereas those with normal FVIII:C showed non-correction, despite equal LA titers.

    “samples with lower FVIII:C showed correction, whereas those with normal FVIII:C showed non-correction.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The cutoff values used for the five indexes are appropriate for the specific APTT reagent and analyzer combination used in this study.

  • The quote only partly states this claim.

    Findingcitation

    The APTT in mixtures was within the normal range for lower LA samples.

    “In fact, the APTT in mixtures was within the normal range [19] (Figure 2B).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [19] Matsuda, 2021, Establishment of activated partial thromboplastin time reference intervals and their clinical utility, using three reagents, Clin Lab Sci J

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

  • May combine two claims.

    Interpretationown data

    Each index had different performance characteristics to LA samples, which were pronounced under low FVIII:C levels in test samples.

    “Each index had different performance characteristics to LA samples, which were pronounced under low FVIII:C levels in test samples.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    The APTT mixing test is useful to delineate the 2 factors, but it potentially becomes a disadvantage in samples that have both factors.

    “The APTT mixing test is useful to delineate the 2 factors, but it potentially becomes a disadvantage in samples that have both factors.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationreasoning

    These findings would be clinically crucial for patients with both LA and lower factor levels.

    “These findings would be clinically crucial for patients with both LA and lower factor levels.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The spiked plasma samples with anti-PS/PT and FVIII deficiency are representative of clinical samples with coexisting LA and coagulation factor deficiency.

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

    Interpretationown data

    Additional lower dilution ratios such as 1/5 might be helpful to reliably and easily read non-parallelism.

    “additional lower dilution ratios such as 1/5 might be helpful to reliably and easily read the non-parallelism (Figure 3).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Only spiked plasma samples were tested.

    “First, only spiked plasma samples were tested.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Lower coagulation factor levels are identified in congenital hemophiliacs (including carriers) and other acquired disorders.

    “Lower coagulation factor levels are identified in congenital hemophiliacs (including carriers), and other acquired disorders [20].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [20] Herbers, 2009, Misleading one-stage coagulation factor assay during rFVIIa treatment in lupus patient, Haemophilia source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Only one combination of reagents and analyzer was used, and the cutoff values in each index may depend on the combination used.

    “Second, we used only one combination of reagents and analyzer; the cutoff values in each index may depend on the combination used.”
    Quote in the paperNot a substantial claim
18 of 18

  • The paper relies on this without support.

    The spiked plasma samples with anti-PS/PT and FVIII deficiency are representative of clinical samples with coexisting LA and coagulation factor deficiency.

    “First, only spiked plasma samples were tested.”
    “These findings would be clinically crucial for patients with both LA and lower factor levels.”

  • The paper relies on this without support.

    The cutoff values used for the five indexes are appropriate for the specific APTT reagent and analyzer combination used in this study.

    “the cutoff values in each index may depend on the combination used.”
    “cutoff values were followed as described in previous studies [1,5,6,15].”
2 of 2

Cites

Flagged first, then by citations

Research works printed but not linked: 1

  1. Matsuda, 2021, Establishment of activated partial thromboplastin time reference intervals and their clinical utility, using three reagents, Clin Lab Sci J, 46, 537

Cited by

Sorted by citations

Related

Authors
Matsuda, M., Hoshiyama, Y., Ogawa, K., Emmi, M., Terai, S., & Moriyama, M.
Affiliations
  • Fukushima Medical University (JP)
  • Niigata University Medical and Dental Hospital (JP)
  • Sekisui Medical (Japan) (JP)
  • Niigata University (JP)
Corresponding author
Matsuda, Masato
ORCID
Venue
Research and Practice in Thrombosis and Haemostasis
Publisher
Elsevier BV
Published
2023-02
Volume
7
Issue
2
Pages
100065
ISSN
2475-0379 (ISSN-L)
DOI
10.1016/j.rpth.2023.100065
PMC
PMC9986643
OpenAlex
W4318953840
Cited by
4
References
23
Citations by year
2026: 1 · 2025: 3
FWCI
0.73
Type
journal article
Language
en
Open access
Licence
Locations
  • Research and Practice in Thrombosis and Haemostasis (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by-nc-nd)
Topics
  • Health Sciences › Medicine › Hematology › Hemophilia Treatment and Research
  • Health Sciences › Medicine › Hematology › Blood Coagulation and Thrombosis Mechanisms
  • Health Sciences › Medicine › Rheumatology › Systemic Lupus Erythematosus Research
Keywords
lupus anticoagulant, hemophilia A, mixing test, activated partial thromboplastin time, coagulation factor deficiency, anticoagulants, factor VIII activity, test performance characteristics

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-08.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-08.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review