Factor IX Assay

Updated: Aug 03, 2021
  • Author: Bishnu Prasad Devkota, MD, MHI, FRCS(Edin), FRCS(Glasg), FACP, FAMIA; Chief Editor: Eric B Staros, MD  more...
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Reference Range

Factor IX (plasma thromboplastin component [PTC]) is produced in the liver. It is a single-chain zymogen with a molecular weight of 57 kd and a plasma half-life of 18-24 hours. It binds effectively to collagen not only in vitro [1] but also, apparently, in vivo, and this may account for the finding that when factor IX is infused into hemophilia B patients, recovery is only 50% of what was expected.

The reference range for factor IX is between 60% and 140% of normal values. [2]



Elevated factor IX concentrations are linked with an increased risk of thromboembolism. The following are associated with increases in factor IX:

  • Pregnancy

  • Use of oral contraceptives

The following are associated with decreases in factor IX [3] :

  • Hemophilia B

  • Liver disease

  • Use of vitamin K antagonists (eg, warfarin) or vitamin K deficiency

  • Amyloidosis

  • Nephrotic syndrome

  • Alloantibodies in hemophilia B patients treated with factor IX infusions

  • Autoantibodies to factor IX in previously healthy individuals (extremely rare)


Collection and Panels

Collection approach and panels for factor IX are as follows:

  • Specimen: Plasma

  • Container: Blue-top vacuum tube

  • Collection method: Routine venipuncture

  • Packing: To comply with Occupational Safety and Health Administration (OSHA) safety standards, samples must be sent in a leakproof sealed container labeled with a biohazard sticker

  • Panels: Quantitative functional assays of coagulation factors




Factor IX is produced in the liver. It circulates as a single-chain zymogen with a molecular weight of 57 kd and a plasma half-life of 18-24 hours. It binds effectively to collagen not only in vitro [1] but also, apparently, in vivo, and this may account for the finding that when factor IX is infused into hemophilia B patients, recovery is only 50% of what was expected. [4] The physiologic relevance of this finding is not yet clear; there is some evidence that factor IX mutants lacking collagen-IV binding may exhibit greater recovery but may be associated with a mild bleeding tendency. [5, 6]

Either activated factor XI (XIa) or the activated factor VII (VIIa)–tissue factor complex may activate factor IX. Activated factor IX (IXa), complexed with activated factor VIII (VIIIa) on a phospholipid membrane surface, activates factor X, and activity normally expressed on the surface of activated platelets. Platelets express a receptor/binding protein for factor IXa that promotes assembly of the IXa/VIIIa complex. [7]

Using hydrogen-deuterium exchange mass spectrometry, a study by Freato et al found that to be stabilized in its enzyme-like form, factor IXa requires not just FVIII binding, but “also interplay between the 220‐loop, N‐terminus, and the substrate binding site.” [8]

Antithrombin (AT) is the primary inhibitor of factor IXa. Such inhibition, though notably slower than the inhibition of thrombin by AT, is enhanced in the presence of heparin.

The gene for factor IX is found on the X chromosome (on the tip of the long arm), [9] indicating that factor IX deficiency is sex-linked. [7] Christmas disease (hemophilia B) is caused by the deficiency of factor IX [10] and is treated with factor IX replacement. Products available on the market include prothrombin complex concentrates (PCCs), which contain prothrombin, factors VII and X, and proteins C and S, in addition to factor IX. PCCs may also contain small amounts of factors VIIa, IXa, and Xa. [11] They may be less expensive than purified factor IX products.

Thromboembolism related to PCC use derives from contamination with activated components. In factor IX-deficient patients with liver dysfunction, inability of the diseased liver to clear the activated factors may lead to thrombosis. [7] Large PCC doses have led to reports of deep vein thrombosis (DVT) and disseminated intravascular coagulation (DIC) in some cases. The preparations now available, however, are less likely to give rise to these complications. As a general rule, if PCCs are to be used for replacement therapy, factor IX levels should not exceed 50% of normal.

Some highly purified factor IX products are derived from human plasma. Although factor IX products synthesized via recombinant DNA technology prevent transmission of prion disease, they are generally associated with lower intravascular recovery of factor IX than purified factor IX products prepared from plasma are. [12] Recombinant factor IX products appear not to give rise to thrombosis. [11] With regard to transmission of hepatitis B and C viruses and HIV, currently available factor IX concentrates are safe, though transmission of these viruses may have occurred in patients treated before 1985. [11]

Inhibitors may develop in factor IX–deficient patients exposed to factor concentrates early in life. In hemophilia B patients undergoing surgical procedures, tranexamic acid may help reduce the risk of perioperative bleeding. [13]

Gene therapy employing adeno-associated viral (AAV) vectors has emerged as a treatment for hemophilia B, with the use of hyperactive factor IX variant R338L (Padua) having been found to ameliorate the problem of liver toxicity linked to high vector doses. A study by Samelson-Jones et al indicated that without factor VIIIa activity, hyperactivity is ablated for recombinant factor IX R338L and for factor IX R338L expressed via the AAV-mediated transgene. [14, 15]


Factor IX testing is indicated in the following situations:

  • Hemophilia B is suspected

  • The clinician needs to differentiate the effects of oral anticoagulants from those of liver disease


Of the more than 100 factor IX gene mutations that have been identified, many cause bleeding diatheses, though some do not give rise to significant symptoms; a few may result in thromboembolism. [16]

Factor IX deficiency does not affect the prothrombin time (PT) appreciably; however, it does prolong the partial thromboplastin time (PTT) when the factor IX level falls to less than 40% of the normal value. [3]

The following factors may decrease the accuracy of the PT [3] :

  • Partial clotting of specimens, resulting from improper mixture of the anticoagulant (3:2 sodium citrate, as per the manufacturer’s blue-top tube)

  • Overfilling or underfilling of test tubes, either of which alters the blood-to-anticoagulant ratio (9:1)

  • Analytical errors (eg, lipemic, icteric, or hemolyzed plasma), which may interfere with photoelectric measuring instruments

In a study of the direct oral anticoagulant (DOAC) rivaroxaban, which can falsely lower factor VIII and IX values in hemophilia testing, Favaloro et al found evidence that DOAC-Stop can more effectively neutralize this effect than can andexanet alfa. Of laboratories reporting abnormal factor VIII and IX levels from a pool of rivaroxaban-spiked plasma, 86% and 100% determined that the values for factors VIII and IX, respectively, had been corrected by DOAC-Stop. In contrast, 59% and 18% of laboratories continued to report abnormal results for factors VIII and IX, respectively, in association with andexanet-alfa treatment. [17]