Mixing study

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Mixing studies are tests performed on blood plasma of patients or test subjects to distinguish factor deficiencies from factor inhibitors, such as lupus anticoagulant, or specific factor inhibitors, such as antibodies directed against factor VIII. [1] 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 (PT), Partial Thromboplastin Time, or sometimes of 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 (PT) or Partial thromboplastin time (PTT) result. [2]

Contents

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%). [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. [4]

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 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 etc.) for 48–72 hours is deficient in Factor VII. Aged plasma is deficient in Factor V & Factor VIIIC. Serum is deficient in factors I, V & VIIIC.

Correction of prothrombin time

Prothrombin time (PT) may be corrected as follows: [5] [6]

Factor Deficiency/AbnormalityProthrombin Time corrected by mixing with
Normal PlasmaAdsorbed PlasmaAged PlasmaCoumarin Plasma
Factor I YesYesYesYes
Factor II YesPartialYesYes
Factor V YesYesNoYes
Factor VII YesNoYesNo
Factor X YesNoYesYes
Anticoagulants NoNoNoNo

Correction of partial thromboplastin time

Partial thromboplastin time (PTT) may be corrected as follows: [5]

Factor Deficiency/AbnormalityPartial Thromboplastin Time corrected by mixing with
Normal PlasmaAdsorbed PlasmaAged Plasma
Factor VIIICYesYesNo
Factor IXYesNoYes
Factor XIYesYesYes

Time-dependent inhibitors

Some inhibitors are time dependent. In other words, it takes time for the antibody to react with and inactivate the added clotting factor. The clotting test performed immediately after the specimens are mixed may show correction because the antibody has not had time to inactivate its target factor. A test performed after the mixture is incubated for 1 to 2 hours at 37°C will show significant prolongation over the clotting time obtained after immediate mixing. Nonspecific inhibitors like the lupus anticoagulant usually are not time dependent; the immediate mixture will show prolongation. Many specific factor inhibitors are time dependent, and the inhibitor will not be detected unless the test is repeated after incubation (factor VIII inhibitors are notorious for this). [7]

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. 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 to digest the heparin (“Hepzyme [8] ”). Also, the patient's history should be checked, especially with regard to anticoagulant use or 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-dependent and nonvitamin K–dependent factors should be considered to rule out vitamin K deficiency, or accidental or surreptitious warfarin ingestion.

Inhibitor

If the mixing study fails to correct, then an inhibitor should be suspected. [9] [10] The most common inhibitor is a nonspecific inhibitor such as a lupus anticoagulant. [9] Perform a test to demonstrate a phospholipid-dependent antibody, such as a platelet neutralization procedure. Spontaneous specific inhibitors against clotting factors occur (i.e. not in hemophiliacs), most often against factor VIII. [11] This can occur in patients with systemic lupus erythematosus, monoclonal gammopathies, other malignancies, during pregnancy and for no apparent reason (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, try porcine factor VIII, activated prothrombin complex concentrate FEIBA (Factor Eight Inhibitor Bypassing Agent), [12] or NovoSeven [13] to stop the bleeding. Prednisone will often lower the titer over time. Intravenous immunoglobulin has been reported to also help but it does not seem to work for hemophiliacs with an inhibitor. Rituximab, cyclophosphamide or other immunosuppressive therapy may be required. [14]

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) [15] and/or the "Chang percentage" (percent correction method) can be used: [16]

Results are: ≤10 is classified as correction, ≥15 indicates presence of an inhibitor, and 11-15 is classified as "indeterminate". [17]

Results are classified as follows: <58% as inhibitor and >70% as correction. [18] >

Alternatively, correction into the reference range can be used to define complete correction. [19]

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. [19]

Related Research Articles

<span class="mw-page-title-main">Coagulation</span> Process of formation of blood clots

Coagulation, also known as clotting, is the process by which blood changes from a liquid to a gel, forming a blood clot. It results in hemostasis, the cessation of blood loss from a damaged vessel, followed by repair. The process of coagulation involves activation, adhesion and aggregation of platelets, as well as deposition and maturation of fibrin.

<span class="mw-page-title-main">Antiphospholipid syndrome</span> Immune disorder leading to increased risk of blood clots

Antiphospholipid syndrome, or antiphospholipid antibody syndrome, is an autoimmune, hypercoagulable state caused by antiphospholipid antibodies. APS can lead to blood clots (thrombosis) in both arteries and veins, pregnancy-related complications, and other symptoms like low platelets, kidney disease, heart disease, and rash. Although the exact etiology of APS is still not clear, genetics is believed to play a key role in the development of the disease.

<span class="mw-page-title-main">Thrombin</span> Enzyme involved in blood coagulation in humans

Prothrombin is encoded in the human by the F2-gene. It is proteolytically cleaved during the clotting process by the prothrombinase enzyme complex to form thrombin.

<span class="mw-page-title-main">Haemophilia C</span> Medical condition

Haemophilia C (also known as plasma thromboplastin antecedent deficiency or Rosenthal syndrome) is a mild form of haemophilia affecting both sexes, due to factor XI deficiency. It predominantly occurs in Ashkenazi Jews. It is the fourth most common coagulation disorder after von Willebrand's disease and haemophilia A and B. In the United States, it is thought to affect 1 in 100,000 of the adult population, making it 10% as common as haemophilia A.

<span class="mw-page-title-main">Prothrombin time</span> Blood test that evaluates clotting

The prothrombin time (PT) – along with its derived measures of prothrombin ratio (PR) and international normalized ratio (INR) – is an assay for evaluating the extrinsic pathway and common pathway of coagulation. This blood test is also called protime INR and PT/INR. They are used to determine the clotting tendency of blood, in such things as the measure of warfarin dosage, liver damage, and vitamin K status. PT measures the following coagulation factors: I (fibrinogen), II (prothrombin), V (proaccelerin), VII (proconvertin), and X.

<span class="mw-page-title-main">Bleeding diathesis</span> High tendency to bleed due to a blood clotting disorder

In medicine (hematology), bleeding diathesis is an unusual susceptibility to bleed (hemorrhage) mostly due to hypocoagulability, in turn caused by a coagulopathy. Therefore, this may result in the reduction of platelets being produced and leads to excessive bleeding. Several types of coagulopathy are distinguished, ranging from mild to lethal. Coagulopathy can be caused by thinning of the skin, such that the skin is weakened and is bruised easily and frequently without any trauma or injury to the body. Also, coagulopathy can be contributed by impaired wound healing or impaired clot formation.

<span class="mw-page-title-main">Partial thromboplastin time</span> Test for coagulation of blood

The partial thromboplastin time (PTT), also known as the activated partial thromboplastin time, is a blood test that characterizes coagulation of the blood. A historical name for this measure is the Kaolin-cephalin clotting time (KCCT), reflecting kaolin and cephalin as materials historically used in the test. Apart from detecting abnormalities in blood clotting, partial thromboplastin time is also used to monitor the treatment effect of heparin, a widely prescribed drug that reduces blood's tendency to clot.

Lupus anticoagulant is an immunoglobulin that binds to phospholipids and proteins associated with the cell membrane. Its name is a partial misnomer, as it is actually a prothrombotic antibody in vivo. The name derives from their properties in vitro, as these antibodies increase coagulation times in laboratory tests such as the activated partial thromboplastin time (aPTT). Investigators speculate that the antibodies interfere with phospholipids used to induce in vitro coagulation. In vivo, the antibodies are thought to interact with platelet membrane phospholipids, increasing adhesion and aggregation of platelets, which accounts for the in vivo prothrombotic characteristics.

<span class="mw-page-title-main">Thrombophilia</span> Abnormality of blood coagulation increasing the risk of blood clotting (thrombosis)

Thrombophilia is an abnormality of blood coagulation that increases the risk of thrombosis. Such abnormalities can be identified in 50% of people who have an episode of thrombosis that was not provoked by other causes. A significant proportion of the population has a detectable thrombophilic abnormality, but most of these develop thrombosis only in the presence of an additional risk factor.

<span class="mw-page-title-main">Factor X</span> Mammalian protein found in Homo sapiens

Coagulation factor X, or Stuart factor, is an enzyme of the coagulation cascade, encoded in humans by F10 gene. It is a serine endopeptidase. Factor X is synthesized in the liver and requires vitamin K for its synthesis.

<span class="mw-page-title-main">Dilute Russell's viper venom time</span> Laboratory test

Dilute Russell's viper venom time (dRVVT) is a laboratory test often used for detection of lupus anticoagulant (LA). It is an assessment of the time for blood to clot in the presence of a diluted amount of venom from Russell's viper, a highly venomous snake native to the Indian subcontinent and named after the herpetologist Patrick Russell.

<span class="mw-page-title-main">Hypoprothrombinemia</span> Medical condition

Hypoprothrombinemia is a rare blood disorder in which a deficiency in immunoreactive prothrombin, produced in the liver, results in an impaired blood clotting reaction, leading to an increased physiological risk for spontaneous bleeding. This condition can be observed in the gastrointestinal system, cranial vault, and superficial integumentary system, affecting both the male and female population. Prothrombin is a critical protein that is involved in the process of hemostasis, as well as illustrating procoagulant activities. This condition is characterized as an autosomal recessive inheritance congenital coagulation disorder affecting 1 per 2,000,000 of the population, worldwide, but is also attributed as acquired.

<span class="mw-page-title-main">Thrombin time</span> Medical diagnostic test

The thrombin time (TT), also known as the thrombin clotting time (TCT), is a blood test that measures the time it takes for a clot to form in the plasma of a blood sample containing anticoagulant, after an excess of thrombin has been added. It is used to diagnose blood coagulation disorders and to assess the effectiveness of fibrinolytic therapy. This test is repeated with pooled plasma from normal patients. The difference in time between the test and the 'normal' indicates an abnormality in the conversion of fibrinogen to fibrin, an insoluble protein.

Prothrombin complex concentrate (PCC), also known as factor IX complex, sold under the brand name Kcentra among others, is a combination medication made up of blood clotting factors II, IX, and X(3-factor PCC) or, when also containing factor VII as does Kcentra, 4-factor PCC. It is used to treat and prevent bleeding in hemophilia B if pure factor IX is not available. It may also be used for reversal of warfarin therapy. It is given by slow injection into a vein. Another product, activated prothrombin complex concentrate or FEIBA, may be used for acquired hemophilia.

Ecarin is an enzyme that is derived from the venom of the Indian saw-scaled viper, Echis carinatus, It is the primary reagent in the Ecarin clotting time test.

Hypercoagulability in pregnancy is the propensity of pregnant women to develop thrombosis. Pregnancy itself is a factor of hypercoagulability, as a physiologically adaptive mechanism to prevent post partum bleeding. However, when combined with an additional underlying hypercoagulable states, the risk of thrombosis or embolism may become substantial.

Clotting time is a general term for the time required for a sample of blood to form a clot, or, in medical terms, coagulate. The term "clotting time" is often used when referring to tests such as the prothrombin time (PT), activated partial thromboplastin time, activated clotting time (ACT), thrombin time (TT), or Reptilase time. These tests are coagulation studies performed to assess the natural clotting ability of a sample of blood. In a clinical setting, healthcare providers will order one of these tests to evaluate a patient's blood for any abnormalities in the time it takes for their blood to clot. Each test involves adding a specific substance to the blood and measuring the time until the blood forms fibrin which is one of the first signs of clotted blood. Each test points to a different component of the clotting sequence which is made up of coagulation factors that help form clots. Abnormal results could be due to a number of reasons including, but, not limited to, deficiency in clotting factors, dysfunction of clotting factors, blood-thinning medications, medication side-effects, platelet deficiency, inherited bleeding or clotting disorders, liver disease, or advanced illness resulting in a medical emergency known as disseminated intravascular coagulation (DIC).

Activated clotting time (ACT), also known as activated coagulation time, is a test of coagulation.

Kaolin clotting time (KCT) is a sensitive test to detect lupus anticoagulants. There is evidence that suggests it is the most sensitive test for detecting lupus anticoagulants. It can also detect factor VIII inhibitors but is sensitive to unfractionated heparin as well.

Acquired haemophilia A (AHA) is a rare but potentially life-threatening bleeding disorder characterized by autoantibodies directed against coagulation factor VIII. These autoantibodies constitute the most common spontaneous inhibitor to any coagulation factor and may induce spontaneous bleeding in patients with no previous history of a bleeding disorder.

References

  1. ↑ Lanzkowsky P (2005-06-06). Manual of Pediatric Hematology and Oncology. Elsevier. ISBN   978-0-08-049731-0.
  2. ↑ Devreese KM (2007). "Interpretation of normal plasma mixing studies in the laboratory diagnosis of lupus anticoagulants". Thrombosis Research. 119 (3): 369–76. doi:10.1016/j.thromres.2006.03.012. PMID   16704874.
  3. ↑ Hoffman R, Benz EJ, Silberstein LE, Heslop H, Anastasi J, Weitz J (2013-01-01). Hematology: Basic Principles and Practice. Elsevier Health Sciences. ISBN   978-1-4377-2928-3.
  4. ↑ Mackie I, Casini A, Pieters M, Pruthi R, Reilly-Stitt C, Suzuki A (February 2024). "International council for standardisation in haematology recommendations on fibrinogen assays, thrombin clotting time and related tests in the investigation of bleeding disorders". Int J Lab Hematol. 46 (1): 20–32. doi:10.1111/ijlh.14201. PMID   37984807.
  5. 1 2 Gupta P, Menon PS, Ramji S, Lodha R (2015-08-31). PG Textbook of Pediatrics: Volume 2: Infections and Systemic Disorders. JP Medical. ISBN   978-93-5152-955-2.
  6. ↑ Med Lab Tech Vol 1, 2/e. Tata McGraw-Hill Education. 2010. ISBN   978-0-07-007659-4.
  7. ↑ Bain BJ, Bates I, Laffan MA (2016-08-11). Dacie and Lewis Practical Haematology E-Book. Elsevier Health Sciences. ISBN   978-0-7020-6925-3.
  8. ↑ "Heparin Neutralization". www.clinlabnavigator.com. 15 January 2017. Retrieved 2018-05-13.
  9. 1 2 "Making Sense of Mixing Studies". George King Bio-Medical, Inc. 2016-01-07. Retrieved 2018-05-13.
  10. ↑ McPherson RA, Pincus MR (2011-09-06). Henry's Clinical Diagnosis and Management by Laboratory Methods E-Book. Elsevier Health Sciences. ISBN   978-1-4557-2684-4.
  11. ↑ Franchini M, Castaman G, Coppola A, Santoro C, Zanon E, Di Minno G, Morfini M, Santagostino E, Rocino A (July 2015). "Acquired inhibitors of clotting factors: AICE recommendations for diagnosis and management". Blood Transfusion. 13 (3): 498–513. doi:10.2450/2015.0141-15. PMC   4614303 . PMID   26192778.
  12. ↑ Lusher JM (2000). "Inhibitor antibodies to factor VIII and factor IX: management". Seminars in Thrombosis and Hemostasis. 26 (2): 179–88. doi:10.1055/s-2000-9821. PMID   10919411. S2CID   43124794.
  13. ↑ Shima M (August 2024). "Current status and future prospects of activated recombinant coagulation factor VIIa, NovoSeven®, in the treatment of haemophilia and rare bleeding disorders". Ann Hematol. 103 (8): 2647–58. doi:10.1007/s00277-023-05287-2. PMC   11283401 . PMID   37391649.
  14. ↑ Rungjirajittranon T, Suwanawiboon B, Nakkinkun Y, Leelakanok N, Kaokunakorn T, Chinthammitr Y, Owattanapanich W, Ruchutrakool T (June 2024). "First-line immunosuppressive therapies for acquired hemophilia A: A 25-year cohort experience and network meta-analysis". Thromb Res. 241: 109067. doi:10.1016/j.thromres.2024.109067. PMID   38970991.
  15. ↑ Favaloro EJ (January 2020). "Coagulation mixing studies: Utility, algorithmic strategies and limitations for lupus anticoagulant testing or follow up of abnormal coagulation tests". Am J Hematol. 95 (1): 117–128. doi:10.1002/ajh.25669. PMID   31674066.
  16. ↑ Baig MA, Swamy KB (2021). "Comparative analysis of chromogenic vs clot.based one stage APTT assay for determination of factor VIII level". Indian J Pathol Microbiol. 64 (1): 123–7. doi: 10.4103/IJPM.IJPM_900_19 . PMID   33433421.
  17. ↑ Rosner, Esther; Pauzner, Rachel; Lusky, Ayala; Modan, Michaela; Many, Amira (1987). "Detection and Quantitative Evaluation of Lupus Circulating Anticoagulant Activity". Thrombosis and Haemostasis. 57 (2): 144–7. doi:10.1055/s-0038-1651083. ISSN   0340-6245. PMID   3110995. S2CID   37633238.
  18. ↑ Chang, Sheng-hsiung; Tillema, Veronica; Scherr, Doris (January 2002). "A "Percent Correction" Formula for Evaluation of Mixing Studies". American Journal of Clinical Pathology. 117 (1): 62–73. doi: 10.1309/rrek-8l6m-d2kc-hwlh . ISSN   0002-9173. PMID   11789732.
  19. 1 2 Chen J, Phillips B, Chandler WL (January 2016). "Evaluation of prothrombin time and activated partial thromboplastin time mixing studies using an estimated factor correction method". Blood Coagul Fibrinolysis. 27 (1): 90–6. doi:10.1097/MBC.0000000000000375. PMID   26397883.
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