Download PDF
Semin Thromb Hemost 2023; 49(06): 571-579
DOI: 10.1055/s-0042-1756196
Review Article

Activated Partial Thromboplastin Time and Prothrombin Time Mixing Studies: Current State of the Art

Dorothy M. Adcock
1   Laboratory Corporation of America, Burlington, North Carolina
,
Gary W. Moore
2   Specialist Haemostasis Unit, Department of Haematology, Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom
3   Department of Natural Sciences, Middlesex University London, London, United Kingdom
,
Silmara de Lima Montalvão
4   Laboratory Hemostasis, Hematology and Hemotherapy Center, University of Campinas (UNICAMP), Campinas-SP, Brazil
,
Geoffrey Kershaw
5   Institute of Haematology, Royal Prince Alfred Hospital, Camperdown, New South Wales, Australia
,
Robert C. Gosselin
6   Davis Health System, Hemostasis and Thrombosis Center, University of California, Sacramento, California
› Author Affiliations
› Further Information
    Permissions and Reprints

Abstract

Mixing studies have long been in the clinical laboratory armamentarium for investigating unexpected, prolonged activated partial thromboplastin time (aPTT) or prothrombin time (PT). The purpose of the mixing study is to identify whether the aPTT/PT prolongation is secondary to a factor deficiency versus an inhibitor, which would present as a “corrected” and “noncorrected” mixing study, respectively. The differentiation between a factor deficiency and inhibitor may likely further direct clinical decisions, including additional diagnostic testing or factor replacement therapy. While aPTT/PT mixing studies are simple tests to perform, there is a lack of standardization for both the testing protocol and the interpretation of what is considered to be a corrected or noncorrected mixing study result. This review will describe the common indications for the mixing test, preanalytic variables that may affect mixing study performance, and describe several methods for interpreting the results of aPTT and PT mixing tests.

Keywords

activated partial thromboplastin time - prothrombin time - mixing study - factor deficiency - factor inhibitor


Publication History

Article published online:
02 September 2022

© 2022. Thieme. All rights reserved.

Thieme Medical Publishers, Inc.
333 Seventh Avenue, 18th Floor, New York, NY 10001, USA

 

  • References

  • 1 Bethel M, Adcock DM. Laboratory evaluation of prolonged APTT and PT. Lab Med 2004; 35 (05) 285-291
    PubMedGoogle Scholar
  • 2 Barbosa ACN, Montalvão SAL, Barbosa KGN. et al. Prolonged APTT of unknown etiology: a systematic evaluation of causes and laboratory resource use in an outpatient hemostasis academic unit. Res Pract Thromb Haemost 2019; 3 (04) 749-757
    CrossrefPubMedGoogle Scholar
  • 3 Adcock DM, Poirier BF. Screening coagulation assays, factor XIII and D-dimer. In: Teruya J. ed. Management of Bleeding Patients. Switzerland: Springer International Publishing; 2016
    CrossrefGoogle Scholar
  • 4 Choi SH, Rambally S, Shen YM. Mixing study for the evaluation of abnormal coagulation test. JAMA 2016; 316 (20) 2146-2147
    CrossrefPubMedGoogle Scholar
  • 5 Favaloro EJ. Coagulation mixing studies: utility, algorithmic strategies and limitations for lupus anticoagulant testing or follow up of abnormal coagulation tests. Am J Hematol 2020; 95 (01) 117-128
    CrossrefPubMedGoogle Scholar
  • 6 National Committee for Clinical Laboratory Standards. One-stage prothrombin time (PT) and activated partial thromboplastin time (APTT) test; approved guidelines. NCCLS Document 1996;28(20):
    PubMedGoogle Scholar
  • 7 Azner E, Rogic D, Meijer P. et al; On Behalf of the Joint Working Group on Postanalytical Phase (WG-POST) of the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) and European Organization for External Quality Assurance Providers in Laboratory Medicine (EQALM). An international study of how laboratories handle and evaluate patient samples after detecting an unexpected APTT prolongation. Clin Chem Lab Med 2015; 53 (01) 1593-1603
    PubMedGoogle Scholar
  • 8 Favaloro EJ, Bonar R, Duncan E. et al. Mis-identification of factor inhibitors by diagnostic haemostasis laboratories: recognition of pitfalls and elucidation of strategies. A follow up to a large multicentre evaluation. Pathology 2007; 39 (05) 504-511
    CrossrefPubMedGoogle Scholar
  • 9 Keeling D, Mackie I, Moore GW, Greer IA, Greaves M. British Committee for Standardization in Haematology. Guidelines on the investigation and management of antiphospholipid syndrome. Br J Haem 2012; 157: 47-58
    CrossrefPubMedGoogle Scholar
  • 10 Devreese KMJ, de Groot PG, de Laat B. et al. Guidance from the Scientific and Standardization Committee for lupus anticoagulant/antiphospholipid antibodies of the International Society on Thrombosis and Haemostasis: Update of the guidelines for lupus anticoagulant detection and interpretation. J Thromb Haemost 2020; 18 (11) 2828-2839
    CrossrefPubMedGoogle Scholar
  • 11 Adcock DM, Hoefner DM, Kottke-Marchant K, Marlar RA, Szamosi DI, Warunek DJ. Collection Transport and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays' Approved Guideline. 5th ed.. Clinical and Laboratory Standards Institute. 2008
    Google Scholar
  • 12 Kitchen S, Adcock DM, Dauer R. et al. International Council for Standardisation in Haematology (ICSH) recommendations for collection of blood samples for coagulation testing. Int J Lab Hematol 2021; 43 (04) 571-580
    CrossrefPubMedGoogle Scholar
  • 13 Baker P, Platton S, Gibson C. et al; British Society for Haematology, Haemostasis and Thrombosis Task Force. Guidelines on the laboratory aspects of assays used in haemostasis and thrombosis. Br J Haematol 2020; 191 (03) 347-362
    CrossrefPubMedGoogle Scholar
  • 14 Adcock DM. Coagulation Assays and Anticoagulant Monitoring. American Society of Hematology Education Program Book. 2012: 460-465
    Google Scholar
  • 15 Adcock DM, Gosselin R. Direct oral anticoagulants (DOACs) in the laboratory: 2015 review. Thromb Res 2015; 136 (01) 7-12
    CrossrefPubMedGoogle Scholar
  • 16 Adcock DM, Favaloro EJ. Pearls and pitfalls in factor inhibitor assays. Int J Lab Hematol 2015; 37 (Suppl. 01) 52-60
    CrossrefPubMedGoogle Scholar
  • 17 Barriere SL, Goldberg MR, Janc JW, Higgins DL, Macy PA, Adcock DM. Effects of telavancin on coagulation test results. Int J Clin Pract 2011; 65 (07) 784-789
    CrossrefPubMedGoogle Scholar
  • 18 Belley A, Robson R, Francis JL. et al. Effects of oritavancin on coagulation tests in the clinical laboratory. Antimicrob Agents Chemother 2017; 61 (02) e01968-e16
    CrossrefPubMedGoogle Scholar
  • 19 Webster PS, Oleson Jr FB, Paterson DL. et al. Interaction of daptomycin with two recombinant thromboplastin reagents leads to falsely prolonged patient prothrombin time/international normalized ratio results. Blood Coagul Fibrinolysis 2008; 19 (01) 32-38
    CrossrefPubMedGoogle Scholar
  • 20 Murphy B, Parrula MC, Perigard CJ, Li J. Artifactual prolongation of activated partial thromboplastin time with PEGylated compounds in silica-based assays. Blood Coagul Fibrinolysis 2014; 25 (08) 876-882
    CrossrefPubMedGoogle Scholar
  • 21 Bethel M, Adcock D, Zalevsky J, Young M. Polyethylene glycol prolongation of aPTT in two biopharmaceuticals. Int J Lab Hematol 2007; 29 (Suppl I ): 69(Abstract)
    PubMedGoogle Scholar
  • 22 Devreese KM, Verfaillie CJ, De Bisschop F, Delanghe JR. Interference of C-reactive protein with clotting times. Clin Chem Lab Med 2015; 53 (05) e141-e145
    CrossrefPubMedGoogle Scholar
  • 23 Fritsma GA, Dembitzer FR, Randhawa A. et al. Recommendations for appropriate activated partial thromboplastin time reagent selection and utilization. Am J Clin Pathol 2012; 137 (06) 904-908
    CrossrefPubMedGoogle Scholar
  • 24 Lawrie AS, Kitchen S, Efthymiou M, Mackie IJ, Machin SJ. Determination of APTT factor sensitivity–the misguiding guideline. Int J Lab Hematol 2013; 35 (06) 652-657
    CrossrefPubMedGoogle Scholar
  • 25 Toulon P, Eloit Y, Smahi M. et al. In vitro sensitivity of different activated partial thromboplastin time reagents to mild clotting factor deficiencies. Int J Lab Hematol 2016; 38 (04) 389-396
    CrossrefPubMedGoogle Scholar
  • 26 Dumoulin EN, Fiers L, Devreese KM. Investigation of sensitivity for coagulation factor deficiency in APTT and PT: how to perform it?. Clin Chem Lab Med 2016; 54 (05) e169-e172
    CrossrefPubMedGoogle Scholar
  • 27 Bowyer A, Kitchen S, Makris M. The responsiveness of different APTT reagents to mild factor VIII, IX and XI deficiencies. Int J Lab Hematol 2011; 33 (02) 154-158
    CrossrefPubMedGoogle Scholar
  • 28 Martinuzzo M, Barrera L, Rodriguez M, D'Adamo MA, López MS, Otaso JC. Do PT and APTT sensitivities to factors' deficiencies calculated by the H47-A2 2008 CLSI guideline reflect the deficiencies found in plasmas from patients?. Int J Lab Hematol 2015; 37 (06) 853-860
    CrossrefPubMedGoogle Scholar
  • 29 CLSI. Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved Guideline. 3rd ed.. CLSI Document C28-A3. Wayne, PA: Clinical and Laboratory Standards Institute; 2008
    Google Scholar
  • 30 Tripodi A. Laboratory testing for lupus anticoagulants: diagnostic criteria and use of screening, mixing, and confirmatory studies. Semin Thromb Hemost 2008; 34 (04) 373-379
    Article in Thieme ConnectPubMedGoogle Scholar
  • 31 Kershaw G, Favaloro EJ. Laboratory identification of factor inhibitors: an update. Pathology 2012; 44 (04) 293-302
    CrossrefPubMedGoogle Scholar
  • 32 Ma AD, Carrizosa D. Acquired factor VIII inhibitors: pathophysiology and treatment. Hematology (Am Soc Hematol Educ Program) 2006; 2006: 432-437
    CrossrefPubMedGoogle Scholar
  • 33 Abdi A, Bordbar MR, Hassan S. et al. Prevalence and incidence of non-neutralizing antibodies in congenital hemophilia A - a systematic review and meta-analysis. Front Immunol 2020; 11: 563
    CrossrefPubMedGoogle Scholar
  • 34 Vivaldi P, Rossetti G, Galli M, Finazzi G. Severe bleeding due to acquired hypoprothrombinemia-lupus anticoagulant syndrome. Case report and review of literature. Haematologica 1997; 82 (03) 345-347
    PubMedGoogle Scholar
  • 35 van den Besselaar AM, Meeuwisse-Braun J. Enzymatic elimination of heparin from plasma for activated partial thromboplastin time and prothrombin time testing. Blood Coagul Fibrinolysis 1993; 4 (04) 635-638
    CrossrefPubMedGoogle Scholar
  • 36 Gosselin RC. Review of coagulation preanalytical variables with update on the effect of direct oral anticoagulants. Int J Lab Hematol 2021; 43 (Suppl. 01) 109-116
    CrossrefPubMedGoogle Scholar
  • 37 Kaczor DA, Bickford NN, Triplett DA. Evaluation of different mixing study reagents and dilution effect in lupus anticoagulant testing. Am J Clin Pathol 1991; 95 (03) 408-411
    CrossrefPubMedGoogle Scholar
  • 38 Kumano O, Moore GW. 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 2017; 2 (01) 105-113
    CrossrefPubMedGoogle Scholar
  • 39 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
    CrossrefPubMedGoogle Scholar
  • 40 Moore GW, Savidge GF. The dilution effect of equal volume mixing studies compromises confirmation of inhibition by lupus anticoagulants even when mixture specific reference ranges are applied. Thromb Res 2006; 118 (04) 523-528
    CrossrefPubMedGoogle Scholar
  • 41 Jin J, Zehnder JL. Prozone effect in the diagnosis of lupus anticoagulant for lupus anticoagulant-hypoprothrombinemia syndrome. Am J Clin Pathol 2016; 146 (02) 262-267
    CrossrefPubMedGoogle Scholar
  • 42 Chang SH, Tillema V, Scherr D. A “percent correction” formula for evaluation of mixing studies. Am J Clin Pathol 2002; 117 (01) 62-73
    CrossrefPubMedGoogle Scholar
  • 43 Rosner E, Pauzner R, Lusky A, Modan M, Many A. Detection and quantitative evaluation of lupus circulating anticoagulant activity. Thromb Haemost 1987; 57 (02) 144-147
    Article in Thieme ConnectPubMedGoogle Scholar
  • 44 Depreter B, Devreese KM. Differences in lupus anticoagulant final conclusion through clotting time or Rosner index for mixing test interpretation. Clin Chem Lab Med 2016; 54 (09) 1511-1516
    PubMedGoogle Scholar
  • 45 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 (10) 1919-1922
    CrossrefPubMedGoogle Scholar
  • 46 Favaloro EJ, Lippi G, Adcock DM. Preanalytical and postanalytical variables: the leading causes of diagnostic error in hemostasis?. Semin Thromb Hemost 2008; 34 (07) 612-634
    Article in Thieme ConnectPubMedGoogle Scholar
  • 47 Favaloro EJ, Adcock DM, Lippi G. Pre-analytical variables in coagulation testing associated with diagnostic errors in hemostasis. Lab Med 2012; 43 (02) DOI: 10.1309/LM749BQETKYPYPVM.
    CrossrefPubMedGoogle Scholar
  • 48 Kitchen S, Adcock DM, Dauer R. et al. International Council for Standardization in Haematology (ICSH) recommendations for processing of blood samples for coagulation testing. Int J Lab Hematol 2021; 43 (06) 1272-1283
    CrossrefPubMedGoogle Scholar
  • 49 Magnette A, Chatelain M, Chatelain B, Ten Cate H, Mullier F. Pre-analytical issues in the haemostasis laboratory: guidance for the clinical laboratories. Thromb J 2016; 14: 49
    CrossrefPubMedGoogle Scholar
  • 50 Gosselin RC, Marlar RA. Preanalytical variables in coagulation testing: setting the stage for accurate results. Semin Thromb Hemost 2019; 45 (05) 433-448
    Article in Thieme ConnectPubMedGoogle Scholar
  • 51 de Lima Montalvão S, Lowe A, Kitchen S. Advantages of external quality assessment - EQA programs. Hemophilia 2022; 1-8 DOI: 10.1111/hae.14562.
    CrossrefPubMedGoogle Scholar
  • 52 Lim EM, Sikaris KA, Gill J. et al. Quality assessment of interpretative commenting in clinical chemistry. Clin Chem 2004; 50 (03) 632-637
    CrossrefPubMedGoogle Scholar
  • 53 Favaloro EJ, Posen J, Ramakrishna R. et al. Factor V inhibitors: rare or not so uncommon? A multi-laboratory investigation. Blood Coagul Fibrinolysis 2004; 15 (08) 637-647
    CrossrefPubMedGoogle Scholar
  • 54 Funk DM, Casciato D. Factor X deficiency. Clin Lab Sci 2010; 23 (03) 131-133
    CrossrefPubMedGoogle Scholar