# Mixing study
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{{Short description|Type of blood plasma screening test}}
'''Mixing studies''' are tests performed on [blood plasma](/source/blood_plasma) of patients or test subjects to distinguish factor deficiencies from [factor inhibitors](/source/Coagulation), such as [lupus anticoagulant](/source/lupus_anticoagulant), or specific factor inhibitors, such as [antibodies](/source/Antibody) directed against [factor VIII](/source/factor_VIII).[{{Cite book|url=https://books.google.com/books?id=hU-xd9xDGxMC&q=coagulation+mixing+studies&pg=PA303|title=Manual of Pediatric Hematology and Oncology|last=Lanzkowsky|first=Philip | name-list-style = vanc |date=2005-06-06|publisher=Elsevier|isbn=978-0-08-049731-0 }}] Mixing studies are screening tests widely performed in coagulation laboratories. The basic purpose of these tests is to determine the cause of prolongation of [Prothrombin Time](/source/Prothrombin_time) (PT), [Partial Thromboplastin Time](/source/Partial_thromboplastin_time), or sometimes of [thrombin time](/source/thrombin_time) (TT). Mixing studies take advantage of the fact that factor levels that are 50 percent of normal should give a normal [Prothrombin time](/source/Prothrombin_time) (PT) or [Partial thromboplastin time](/source/Partial_thromboplastin_time) (PTT) result.[{{cite journal | vauthors = Devreese KM | title = Interpretation of normal plasma mixing studies in the laboratory diagnosis of lupus anticoagulants | journal = Thrombosis Research | volume = 119 | issue = 3 | pages = 369–76 | year = 2007 | pmid = 16704874 | doi = 10.1016/j.thromres.2006.03.012 | url = https://biblio.ugent.be/publication/742294/file/763496 | url-access = subscription }}]
==Test method==
Fresh normal plasma has all the blood coagulation factors with normal levels.
If the problem is a simple factor deficiency, mixing the patient plasma 1:1 with plasma that contains 100% of the normal factor level results in a level ≥50% in the mixture (say the patient has an activity of 0%; the average of 100% + 0% = 50%).[{{cite book | url = https://books.google.com/books?id=a1estSuaQ6kC&q=coagulation+mixing+studies&pg=PA1863 |title=Hematology: Basic Principles and Practice|last1=Hoffman|first1=Ronald |first2=Edward J. | last2 = Benz|last3=Silberstein|first3=Leslie E.|last4=Heslop|first4=Helen|last5=Anastasi|first5=John|last6=Weitz|first6=Jeffrey | name-list-style = vanc |date=2013-01-01|publisher=Elsevier Health Sciences|isbn=978-1-4377-2928-3}}] The PT or PTT will be normal (the mixing study shows correction). Correction with mixing indicates factor deficiency. Failure to correct with mixing indicates an inhibitor. Performing a thrombin time on the test plasma can provide useful additional information for the interpretation of mixing tests, such as by demonstrating the presence of anticoagulants, hypofibrinogenemia or dysfibrinogenemia.[{{cite journal |vauthors=Mackie I, Casini A, Pieters M, Pruthi R, Reilly-Stitt C, Suzuki A |title=International council for standardisation in haematology recommendations on fibrinogen assays, thrombin clotting time and related tests in the investigation of bleeding disorders |journal=Int J Lab Hematol |volume=46 |issue=1 |pages=20–32 |date=February 2024 |pmid=37984807 |doi=10.1111/ijlh.14201 }}]
==Adsorbed Plasma and Aged Plasma==
Factor deficient plasmas (Adsorbed Plasma and Aged Plasma, etc.) have been used historically in mixing studies. Plasma with known factor deficiencies are commercially available but are very expensive, so they have been prepared in the laboratory and used for mixing tests. Adsorbed plasma or plasma from patients on oral anticoagulants ([Warfarin](/source/Warfarin) etc.) for a week or more is deficient in Factor II, Factor VII, Factor IX, and Factor X. Plasma from patients on oral anticoagulants ([Warfarin](/source/Warfarin) etc.) for 48–72 hours is deficient in [Factor VII](/source/Factor_VII). Aged plasma is deficient in [Factor V](/source/Factor_V) & [Factor VIII](/source/Factor_VIII)C. Serum is deficient in factors I, V & VIIIC.
=== Correction of prothrombin time ===
[Prothrombin time](/source/Prothrombin_time) (PT) may be corrected as follows:[{{Cite book|url=https://books.google.com/books?id=krlEDwAAQBAJ&q=adsorbed+plasma+aged+plasma+correction&pg=PA1600|title=PG Textbook of Pediatrics: Volume 2: Infections and Systemic Disorders|last1=Gupta|first1=Piyush|last2=Menon|first2=P. S. N.|last3=Ramji|first3=Siddarth|last4=Lodha|first4=Rakesh | name-list-style = vanc |date=2015-08-31|publisher=JP Medical |isbn=978-93-5152-955-2 }}][{{cite book |url = https://books.google.com/books?id=z_8hBAAAQBAJ&q=adsorbed+plasma+aged+plasma+correction&pg=PA344 | title=Med Lab Tech Vol 1, 2/e|date=2010|publisher=Tata McGraw-Hill Education|isbn=978-0-07-007659-4}}]
{| class="wikitable"
| rowspan="2" |'''Factor Deficiency/Abnormality'''
| colspan="4" |'''Prothrombin Time corrected by mixing with'''
|-
|'''Normal Plasma'''
|'''Adsorbed Plasma'''
|'''Aged Plasma'''
|'''Coumarin Plasma'''
|-
|[Factor I](/source/Factor_I_Deficiency)
|style="background: PaleGreen;" | Yes
|style="background: PaleGreen;" |Yes
|style="background: PaleGreen;" |Yes
|style="background: PaleGreen;" |Yes
|-
|[Factor II](/source/Thrombin)
|style="background: PaleGreen;" |Yes
|style="background: AntiqueWhite;" |Partial
|style="background: PaleGreen;" |Yes
|style="background: PaleGreen;" |Yes
|-
|[Factor V](/source/Factor_V)
|style="background: PaleGreen;" |Yes
|style="background: PaleGreen;" |Yes
|style="background: Salmon;" |No
|style="background: PaleGreen;" |Yes
|-
|[Factor VII](/source/Factor_VII)
|style="background: PaleGreen;" |Yes
|style="background: Salmon;" |No
|style="background: PaleGreen;" |Yes
|style="background: Salmon;" |No
|-
|[Factor X](/source/Factor_X)
|style="background: PaleGreen;" |Yes
|style="background: Salmon;" |No
|style="background: PaleGreen;" |Yes
|style="background: PaleGreen;" |Yes
|-
|[Anticoagulant](/source/Anticoagulant)s
|style="background: Salmon;" |No
|style="background: Salmon;" |No
|style="background: Salmon;" |No
|style="background: Salmon;" |No
|}
=== Correction of partial thromboplastin time ===
[Partial thromboplastin time](/source/Partial_thromboplastin_time) (PTT) may be corrected as follows:[{{Cite book|url=https://books.google.com/books?id=rEPUDAAAQBAJ&q=adsorbed+plasma+aged+plasma+correction&pg=PA385|title=Dacie and Lewis Practical Haematology E-Book|last1=Bain|first1=Barbara J.|last2=Bates|first2=Imelda|last3=Laffan|first3=Mike A. | name-list-style = vanc |date=2016-08-11|publisher=Elsevier Health Sciences|isbn=978-0-7020-6925-3}}]
==Abnormal coagulation test results==
A common problem is an unexplained increase in the PT and/or PTT. If this is observed, the test should be repeated with a fresh sample. Another consideration is [heparin](/source/heparin). It is possible that the blood sample was mistakenly drawn though a running line. Interference by heparin can be detected by absorbing the heparin with a resin ("Heparsorb") or by using an [enzyme](/source/enzyme) to digest the heparin ("Hepzyme[{{Cite web|url=http://www.clinlabnavigator.com/heparin-neutralization.html|title=Heparin Neutralization|website=www.clinlabnavigator.com |date=15 January 2017 |access-date=2018-05-13}}]"). Also, the patient's [history](/source/Medical_history) should be checked, especially with regard to anticoagulant use or [liver disease](/source/liver_disease). Provided that the abnormal result is reproduced on a fresh specimen and there is no obvious explanation from the history, a mixing study should be performed. If the mixing study shows correction and no prolongation with incubation, factor deficiency should be looked for, starting with VIII and IX. [Vitamin K](/source/Vitamin_K)-dependent and nonvitamin K–dependent factors should be considered to rule out vitamin K deficiency, or accidental or surreptitious [warfarin](/source/warfarin) ingestion.
==Inhibitor==
If the mixing study fails to correct, then an inhibitor should be suspected.[{{cite web | url = https://kingbiomed.com/blog/making-sense-of-mixing-studies/|title=Making Sense of Mixing Studies |date=2016-01-07|work=George King Bio-Medical, Inc.|access-date=2018-05-13 }}][{{Cite book|url=https://books.google.com/books?id=-97J-8Zh2hkC&q=If+the+mixing+study+fails+to+correct%2C+then+you+need+to+think+about+an+inhibitor&pg=PA794|title=Henry's Clinical Diagnosis and Management by Laboratory Methods E-Book|last1=McPherson|first1=Richard A.|last2=Pincus|first2=Matthew R. | name-list-style = vanc |date=2011-09-06|publisher=Elsevier Health Sciences|isbn=978-1-4557-2684-4}}] The most common inhibitor is a nonspecific inhibitor such as a [lupus anticoagulant](/source/lupus_anticoagulant).[{{cite journal | vauthors = Franchini M, Castaman G, Coppola A, Santoro C, Zanon E, Di Minno G, Morfini M, Santagostino E, Rocino A | title = Acquired inhibitors of clotting factors: AICE recommendations for diagnosis and management | journal = Blood Transfusion | volume = 13 | issue = 3 | pages = 498–513 | date = July 2015 | pmid = 26192778 | pmc = 4614303 | doi = 10.2450/2015.0141-15 }}] This can occur in patients with [systemic lupus erythematosus](/source/systemic_lupus_erythematosus), [monoclonal gammopathies](/source/Monoclonal_gammopathy_of_undetermined_significance), other [malignancies](/source/Malignancy), during [pregnancy](/source/pregnancy) and for no apparent reason ([idiopathic](/source/idiopathic)). These patients can have devastating bleeding. The thing to do is identify the specific factor involved and find out how high the titer is. If the patient has a low titer inhibitor, try to overwhelm it with high doses of the factor. If the patient has a high titer antibody against [factor VIII](/source/factor_VIII), try [porcine factor VIII](/source/porcine_factor_VIII), activated prothrombin complex concentrate [FEIBA](/source/FEIBA) (Factor Eight Inhibitor Bypassing Agent),[{{cite journal | vauthors = Lusher JM | title = Inhibitor antibodies to factor VIII and factor IX: management | journal = Seminars in Thrombosis and Hemostasis | volume = 26 | issue = 2 | pages = 179–88 | date = 2000 | pmid = 10919411 | doi = 10.1055/s-2000-9821 | s2cid = 43124794 }}] or [NovoSeven](/source/NovoSeven)[{{cite journal |vauthors=Shima M |title=Current status and future prospects of activated recombinant coagulation factor VIIa, NovoSeven®, in the treatment of haemophilia and rare bleeding disorders |journal=Ann Hematol |volume=103 |issue=8 |pages=2647–58 |date=August 2024 |pmid=37391649 |pmc=11283401 |doi=10.1007/s00277-023-05287-2 }}] to stop the bleeding. [Prednisone](/source/Prednisone) will often lower the titer over time. [Intravenous immunoglobulin](/source/Intravenous_immunoglobulin) has been reported to also help but it does not seem to work for hemophiliacs with an inhibitor. [Rituximab](/source/Rituximab), [cyclophosphamide](/source/cyclophosphamide) or other immunosuppressive therapy may be required.[{{cite journal |vauthors=Rungjirajittranon T, Suwanawiboon B, Nakkinkun Y, Leelakanok N, Kaokunakorn T, Chinthammitr Y, Owattanapanich W, Ruchutrakool T |title=First-line immunosuppressive therapies for acquired hemophilia A: A 25-year cohort experience and network meta-analysis |journal=Thromb Res |volume=241 |issue= |article-number=109067 |date=June 2024 |pmid=38970991 |doi=10.1016/j.thromres.2024.109067 |doi-access=free }}]
=== Assessing correction of mixing study ===
In order to provide specific cutoffs to distinguish an inhibitor defect from a factor deficiency, the "Rosner index" (index of circulating anticoagulant)[{{cite journal |vauthors=Favaloro EJ |title=Coagulation mixing studies: Utility, algorithmic strategies and limitations for lupus anticoagulant testing or follow up of abnormal coagulation tests |journal=Am J Hematol |volume=95 |issue=1 |pages=117–128 |date=January 2020 |pmid=31674066 |doi=10.1002/ajh.25669 }}] and/or the "Chang percentage" (percent correction method) can be used:[{{cite journal| author=Baig MA, Swamy KB| title=Comparative analysis of chromogenic vs clot.based one stage APTT assay for determination of factor VIII level. | journal=Indian J Pathol Microbiol | year= 2021 | volume= 64 | issue= 1 | pages= 123–7 | pmid=33433421 |doi=10.4103/IJPM.IJPM_900_19 |doi-access=free| pmc= }}]
Results are: ≤10 is classified as correction, ≥15 indicates presence of an inhibitor, and 11-15 is classified as "indeterminate".[{{Cite journal |last1=Rosner |first1=Esther |last2=Pauzner |first2=Rachel |last3=Lusky |first3=Ayala |last4=Modan |first4=Michaela |last5=Many |first5=Amira |date=1987 |title=Detection and Quantitative Evaluation of Lupus Circulating Anticoagulant Activity |journal=Thrombosis and Haemostasis |volume=57 |issue=2 |pages=144–7 |doi=10.1055/s-0038-1651083 |pmid=3110995 |s2cid=37633238 |issn=0340-6245}}]
Results are classified as follows: <58% as inhibitor and >70% as correction.[{{Cite journal |last1=Chang |first1=Sheng-hsiung |last2=Tillema |first2=Veronica |last3=Scherr |first3=Doris |date=January 2002 |title=A "Percent Correction" Formula for Evaluation of Mixing Studies |journal=American Journal of Clinical Pathology |volume=117 |issue=1 |pages=62–73 |doi=10.1309/rrek-8l6m-d2kc-hwlh |pmid=11789732 |issn=0002-9173|doi-access=free }}]
Alternatively, correction into the reference range can be used to define complete correction.[{{cite journal |vauthors=Chen J, Phillips B, Chandler WL |title=Evaluation of prothrombin time and activated partial thromboplastin time mixing studies using an estimated factor correction method |journal=Blood Coagul Fibrinolysis |volume=27 |issue=1 |pages=90–6 |date=January 2016 |pmid=26397883 |doi=10.1097/MBC.0000000000000375 }}]
A fourth method is known as Estimated Factor Correction (EFC). This method involves four steps. First, determine the most likely factor suspected to be deficient, based on PT, aPTT, and clinical history. Next, choose the appropriate curve — single factor deficiency, vitamin K-dependent factor deficient, or all factor-deficient. Use this curve to estimate the factor level in the patient sample. Then, predict the factor level and PT or aPTT that will occur after 1:1 mix in case of deficiency. Finally, compare the actual mix results with the predicted results for deficiency.