Mixing test
Encyclopedia
Mixing studies are tests performed on blood plasma
used to distinguish factor deficiencies from factor inhibitors, such as lupus anticoagulant
, or specific factor inhibitors, such as antibodies
directed against factor VIII
. 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.
that inactivates the added clotting factor, the resulting factor level will be low and the clotting test will be prolonged (fails to correct). Therefore, correction with mixing indicates factor deficiency; failure to correct indicates an inhibitor.
. 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”). Also, the patient's history
should be checked: Is the patient on any anticoagulants? Does the patient have liver disease
? Provided that the abnormal result is reproduced on a good specimen and there is no obvious explanation from the history, the next thing to do is a mixing study. If the mixing study shows correction and no prolongation with incubation, factor assays will need to be done to look for factor deficiency, starting with VIII and IX since they are the most common deficiencies. It is useful to do a few vitamin K–dependent
and a few nonvitamin K–dependent factors to be sure that the problem is not accidental or surreptitious warfarin
ingestion.
neutralization procedure. Spontaneous specific inhibitors against clotting factors occur (i.e. not in hemophiliacs
), most often against factor VIII. This can occur in patients with systemic lupus
, [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 or prothrombin
complex concentrates 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.
Blood plasma
Blood plasma is the straw-colored liquid component of blood in which the blood cells in whole blood are normally suspended. It makes up about 55% of the total blood volume. It is the intravascular fluid part of extracellular fluid...
used to distinguish factor deficiencies from factor inhibitors, such as lupus anticoagulant
Lupus anticoagulant
Lupus anticoagulant is an immunoglobulin that binds to phospholipids and proteins associated with the cell membrane. It is important to note that this disease name is actually a misnomer...
, or specific factor inhibitors, such as antibodies
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
directed against factor VIII
Factor VIII
Factor VIII is an essential blood clotting factor also known as anti-hemophilic factor . In humans, Factor VIII is encoded by the F8 gene...
. Mixing studies take advantage of the fact that factor levels that are 50 percent of normal should give a normal Prothrombin time
Prothrombin time
The prothrombin time and its derived measures of prothrombin ratio and international normalized ratio are measures of the extrinsic pathway of coagulation. This test is also called "ProTime INR" and "INR PT". They are used to determine the clotting tendency of blood, in the measure of warfarin...
(PT) or Partial thromboplastin time
Partial thromboplastin time
The partial thromboplastin time or activated partial thromboplastin time is a performance indicator measuring the efficacy of both the "intrinsic" and the common coagulation pathways...
(PTT) result.
Test method
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%). The PT or PTT will be normal (the mixing study shows correction). However, if there is an inhibitorEnzyme inhibitor
An enzyme inhibitor is a molecule that binds to enzymes and decreases their activity. Since blocking an enzyme's activity can kill a pathogen or correct a metabolic imbalance, many drugs are enzyme inhibitors. They are also used as herbicides and pesticides...
that inactivates the added clotting factor, the resulting factor level will be low and the clotting test will be prolonged (fails to correct). Therefore, correction with mixing indicates factor deficiency; failure to correct indicates an inhibitor.
Time-dependent inhibitors
Unfortunately, it is not as simple as that. 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 the added factor. A test performed after the mixture is incubated for 2 hours at 37°C will show prolongation. 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).Abnormal coagulation test results
A common problem is an unexplained increase in the PT and/or PTT. The first thing to do is get a fresh sample and repeat the test. Another consideration is heparinHeparin
Heparin , also known as unfractionated heparin, a highly sulfated glycosaminoglycan, is widely used as an injectable anticoagulant, and has the highest negative charge density of any known biological molecule...
. 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
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
to digest the heparin (“Hepzyme”). Also, the patient's history
Medical history
The medical history or anamnesis of a patient is information gained by a physician by asking specific questions, either of the patient or of other people who know the person and can give suitable information , with the aim of obtaining information useful in formulating a diagnosis and providing...
should be checked: Is the patient on any anticoagulants? Does the patient have liver disease
Liver disease
Liver disease is a broad term describing any single number of diseases affecting the liver.-Diseases:* Hepatitis, inflammation of the liver, caused mainly by various viruses but also by some poisons , autoimmunity or hereditary conditions...
? Provided that the abnormal result is reproduced on a good specimen and there is no obvious explanation from the history, the next thing to do is a mixing study. If the mixing study shows correction and no prolongation with incubation, factor assays will need to be done to look for factor deficiency, starting with VIII and IX since they are the most common deficiencies. It is useful to do a few vitamin K–dependent
Vitamin K
Vitamin K is a group of structurally similar, fat soluble vitamins that are needed for the posttranslational modification of certain proteins required for blood coagulation and in metabolic pathways in bone and other tissue. They are 2-methyl-1,4-naphthoquinone derivatives...
and a few nonvitamin K–dependent factors to be sure that the problem is not accidental or surreptitious warfarin
Warfarin
Warfarin is an anticoagulant. It is most likely to be the drug popularly referred to as a "blood thinner," yet this is a misnomer, since it does not affect the thickness or viscosity of blood...
ingestion.
Factor deficiencies
If the mixing study fails to correct, then you need to think about an inhibitor. The most common inhibitor is a nonspecific inhibitor such as a lupus anticoagulant. Perform a test to demonstrate a phospholipid-dependent antibody, such as a plateletPlatelet
Platelets, or thrombocytes , are small,irregularly shaped clear cell fragments , 2–3 µm in diameter, which are derived from fragmentation of precursor megakaryocytes. The average lifespan of a platelet is normally just 5 to 9 days...
neutralization procedure. Spontaneous specific inhibitors against clotting factors occur (i.e. not in hemophiliacs
Haemophilia
Haemophilia is a group of hereditary genetic disorders that impair the body's ability to control blood clotting or coagulation, which is used to stop bleeding when a blood vessel is broken. Haemophilia A is the most common form of the disorder, present in about 1 in 5,000–10,000 male births...
), most often against factor VIII. This can occur in patients with systemic lupus
Systemic lupus erythematosus
Systemic lupus erythematosus , often abbreviated to SLE or lupus, is a systemic autoimmune disease that can affect any part of the body. As occurs in other autoimmune diseases, the immune system attacks the body's cells and tissue, resulting in inflammation and tissue damage...
, [monoclonal gammopathies
Monoclonal gammopathy of undetermined significance
Monoclonal gammopathy of undetermined significance , formerly benign monoclonal gammopathy, is a condition in which a paraprotein is found in the blood during standard laboratory tests...
, other malignancies, during pregnancy
Pregnancy
Pregnancy refers to the fertilization and development of one or more offspring, known as a fetus or embryo, in a woman's uterus. In a pregnancy, there can be multiple gestations, as in the case of twins or triplets...
and for no apparent reason (idiopathic
Idiopathic
Idiopathic is an adjective used primarily in medicine meaning arising spontaneously or from an obscure or unknown cause. From Greek ἴδιος, idios + πάθος, pathos , it means approximately "a disease of its own kind". It is technically a term from nosology, the classification of disease...
). 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
Factor VIII
Factor VIII is an essential blood clotting factor also known as anti-hemophilic factor . In humans, Factor VIII is encoded by the F8 gene...
, try porcine factor VIII or prothrombin
Thrombin
Thrombin is a "trypsin-like" serine protease protein that in humans is encoded by the F2 gene. Prothrombin is proteolytically cleaved to form thrombin in the first step of the coagulation cascade, which ultimately results in the stemming of blood loss...
complex concentrates to stop the bleeding. Prednisone
Prednisone
Prednisone is a synthetic corticosteroid drug that is particularly effective as an immunosuppressant drug. It is used to treat certain inflammatory diseases and some types of cancer, but has significant adverse effects...
will often lower the titer over time. Intravenous immunoglobulin
Intravenous immunoglobulin
Intravenous immunoglobulin is a blood product administered intravenously. It contains the pooled IgG extracted from the plasma of over one thousand blood donors. IVIG's effects last between 2 weeks and 3 months...
has been reported to also help but it does not seem to work for hemophiliacs with an inhibitor.