Chromogenic Factor VIII and IX Assays: Impact on Diagnosis and Management of Hemophilia

Expires: March 28, 2021



Image of Rajiv K. Pruthi, M.B.B.S.Presenter

Rajiv Pruthi, M.B.B.S., is the Co-Director of the Special Coagulation Laboratory and Director of the Comprehensive Hemophilia Center at Mayo Clinic in Rochester, Minnesota. He holds the academic rank of Associate Professor of Medicine.


Contact us:

Transcript and References


Hi, I’m Matt Binnicker, the Director of Clinical Virology and Vice Chair of Practice in the Department of Laboratory Medicine and Pathology at Mayo Clinic. In this month’s “Hot Topic” my colleague, Dr. Rajiv Pruthi, will discuss different types of hemophilia along with their pathologic basis. He will also cover various types of factor assays such as one-stage and chromogenic factor assays for diagnosis and their role in management of hemophilia. I hope you enjoy this month’s Hot Topic, and I want to personally thank you for allowing Mayo Clinic the opportunity to be a partner in your patients’ health care.

Thank you very much for the introduction. I will be presenting information on chromogenic factor VIII and IX assays and their impact on diagnosis and management of hemophilia.


I have no relevant disclosures.


At the end of this presentation, the audience will be able to recall the types of bleeding disorders, state the types of hemophilia, review the different types of one-stage and chromogenic factor VIII and IX activity assays, and discuss the roles and limitations of factor assays in the diagnosis and management of hemophilia.

Congenital Bleeding Disorders: Types and Approximate Frequency

Congenital bleeding disorders may be categorized into coagulation factor deficiencies and platelet disorders. The most common coagulation factor deficiency-related bleeding disorder is von Willebrand disease, a deficiency of von Willebrand factor. This affects approximately 1% of the general population. Hemophilia A affects approximately 1 in 10,000 and hemophilia B affects approximately 1 in 30,000 live male births. Hemophilia C, which is factor XI deficiency, affects 1 in 100,000 of the U.S. general population but up to 8% of individuals of Ashkenazi Jewish descent. The prevalence of platelet disorders is not accurately quantified; they are rare but are also likely under diagnosed.

Hemophilia A and B: X-Linked Recessive

Hemophilia A and hemophilia B are X-linked recessive bleeding disorders. This means that males are affected and females are asymptomatic carriers, about 90% of whom do not have bleeding symptoms. Approximately 10% do have low enough factor levels and experience bleeding. Severity of hemophilia is based on baseline coagulation factor activity levels. Severe hemophilia patients have a factor VIII or factor IX activity level of less than 1%, whereas in moderate hemophilia the activity level ranges from 1 to 5% and with mild hemophilia it is equal to or greater than 6%.

Role of Measurement of Factor Activity Levels

The indications for measurement of coagulation factor activity assays can fall under three categories. These include diagnostic, prognostic, and therapeutic indications. Since hemophilia A and B are clinically indistinguishable from each other, it is important to make an accurate diagnosis of hemophilia based on coagulation factor assays. In addition, based upon baseline factor activity level, the patient is classified into mild, moderate, or severe hemophilia. Patients with severe hemophilia are at a higher risk of spontaneous hemorrhage and therefore are managed with prophylactic infusions of coagulation factor concentrates to prevent bleeding. This is also known as prophylaxis. Finally, accurate measurement of coagulation factor levels after infusion of clotting factor concentrates is important in order to be able to provide cost-effective factor replacement therapy.

One-Stage Assay

Next we will review the one-stage and chromogenic assays. The one-stage assay is the most commonly used assay in the United States. Patient plasma, which contains all the clotting factors except the factor that is being tested (like factor VIII, which may be reduced or absent), is initially mixed with factor VIII-deficient plasma. Coagulation is initiated by addition of a contact activator, and time to clot formation is measured. The end point is typically detected using either optical or mechanical and point detection systems. The contact activator may be silica, kaolin, or ellagic acid based.

Chromogenic Factor VIII Assay

In the chromogenic factor VIII assay, also known as the two-stage assay, the main rate-limiting step is the factor VIII level present in the test plasma. In stage one, reagents containing factor X and activated factor IX, with or without thrombin, are added to test plasma. This mixture activates factor X. In the second stage of this assay, the activated factor X cleaves a chromogenic substrate. The color generated is proportional to factor VIII activity in the patient plasma and is read photometrically. It is important to note that factor X in the reagent can be of human origin or bovine origin, and this makes a difference in whether the particular chromogenic assay can be used to measure factor VIII inhibitors in patients who are treated with a drug called emicizumab (HEMLIBRA). We will address this in a subsequent slide.

Chromogenic Factor IX Assay

In the chromogenic factor IX assay, the rate-limiting step is the factor IX activity present in the test plasma. In the first stage, reagents containing calcium, phospholipid, and activated factors XI and VIII are added to the test plasma. This generates activated factor IX and subsequently activates factor X and cleaves the chromogenic substrate, which is photometrically measured as in the chromogenic factor VII assay.

Comparison 1

Some features to compare the one-stage assay with the chromogenic assay are shown on this slide. The reference cited provides a very good discussion of these assays. There are advantages and disadvantages to each assay, some of which are listed here. One major disadvantage of the one-stage assay is the high intra- laboratory variation as demonstrated in the proficiency-testing exercises. There is less variation with the chromogenic assay.

Standard of Care for Severe Hemophilia

The standard of care for management of hemophilia in general and severe hemophilia in particular is to provide prophylaxis. This means regular intravenous administration of coagulation factor concentrates in a preventative fashion to prevent bleeding. Given the different half-lives of factor VIII and factor IX, the frequency of infusions may vary from three per week for factor VIII and twice weekly for factor IX. This frequency has been reduced to variable degrees with the availability of modified recombinant factor concentrates to extend their half-lives. Whether one uses the standard half-life or the extended half-life concentrate, the goal is to maintain trough factor levels of greater than 1%. In order to achieve this goal, post-infusion pharmacokinetic studies are typically performed by serial measurements of factor levels after infusion of a dose of factor concentrate. The infusion doses are then adjusted to be able to achieve a through factor level of greater than 1%.

Case History #1: New Modified rFIXFc

Next we will discuss a case illustrating the importance of the type of contact activator used in the one-stage assay and its effect on measurement of factor IX. A young patient with hemophilia B was initiated on an extended half-life factor IX concentrate. Following the standardized practice, a pharmacokinetic study was performed to determine the optimal dose and frequency of factor infusion. In this patient, the trough or pre-infusion factor IX was 5%, and a sample obtained approximately one hour after infusion was 80%.

The patient was retested using a different laboratory and therefore different reagent set. He had experienced no unexpected bleeding complications while on prophylactic infusions of his factor IX concentrate.

The laboratory data on repeat testing looked quite different. The baseline factor IX was less than 1%, and one hour after infusion of the same dose of his extended half-life factor IX concentrate, the data revealed a suboptimal peak.

Based on those results, the local hemophilia center planned to increase the dose of factor IX concentrate. However, the center communicated with our laboratory prior to doing so. It was discovered that the contact activator used in their laboratory was kaolin based, which has been shown to significantly underestimate the true factor IX activity level for this particular extended half-life concentrate. Prior to increasing the dose of the patient's factor IX concentrate, we advised that the factor assays be repeated using the appropriate silica-based contact activator, and this confirmed the original pharmacokinetic study results. Therefore, the dosage of the factor IX concentrate did not need to be increased. This case demonstrates one of the disadvantages with one-stage assays, due to different contact activators. It would be important for laboratories to know which contact activator is appropriate for which extended half-life factor concentrate. This is likely a complex task since laboratories do not always know which factor concentrate a patient is being treated with. Using a chromogenic factor assay will likely overcome this limitation since the chromogenic assays perform well in the large majority of the factor concentrates.

Case History #2: Pharmacokinetic Study with New Modified Recombinant Factor VIII (rFVIII)

In this second illustrative case, a similar pharmacokinetic study was done on a patient with severe hemophilia A, who had just switched to a new modified rFVIII. The patient received approximately 30 units/kilogram of the new modified rFVIII. The dose was calculated to achieve a post-infusion target of approximately 60%. However, when measured with the one-stage assay, the post-infusion level was only 30%.

Case History #2: Pharmacokinetic Study with New Modified rFVIII

When the same plasma sample was used to measure factor VIII using the chromogenic factor VIII assay, results were markedly different, and indeed the dosage was accurately calculated to achieve the intended target. So for this particular modified rFVIII concentrate, the chromogenic assay was more accurate than the one-stage assay.

Consequences/Risks of Inaccurate Post-Infusion Coagulation Factor Measurements

The potential consequences of inaccurate measurement of clotting factor concentrates include underestimation of factor levels (which may lead to overdosing of the factor concentrate), increased cost, and increased risk of thrombotic complications. On the other hand, overestimation of true factor level may result in potential underdosing of the factor concentrate and increasing the risk of bleeding and accompanying morbidity.

Effect of Inaccurate Measurement of Coagulation Factor Levels on the Diagnosis of Hemophilia

Next we will talk about the effect of inaccurate measurement of coagulation factor levels on the diagnosis of hemophilia.

Discrepancy between Factor VIII and Factor IX Activity Assays: Potential for Missed Diagnosis2,3,4

For patients with severe hemophilia A, there is generally no discrepancy between the one-stage and chromogenic factor VIII assays. However, a discrepancy between the one-stage and chromogenic factor VIII and IX assays in patients with non-severe hemophilia has recently been observed. Up to 30% of patients with hemophilia A may have a discordant result. Most of the time, the one-stage assay result is higher than the chromogenic result, but it has been observed that the chromogenic assay result may also be higher. In selected cases, either the one-stage assay or chromogenic assay result may be completely normal, potentially missing the diagnosis of hemophilia. This discrepancy was observed in patients with very specific factor VIII gene mutations. More recently, this phenomenon was described in patients with hemophilia B as well.

Emicizumab and Coagulation Factor VIII Assays

Next we discuss how emicizumab, a newly FDA-approved medication for use in patients with hemophilia A with or without inhibitors, affects the factor VIII assays.

This cartoon demonstrates the mechanism of action of emicizumab. As shown in the upper panel, normally, factor IX needs to activate factor X for optimal clot formation. The role of factor VIII is to help this process, and thus it acts as a cofactor for this activation step. In patients with hemophilia who have a deficiency factor VIII or have developed inhibitor antibodies against factor VIII, activation of factor X is either delayed or completely inhibited. In order to bypass this block, a medication called emicizumab, which is a bispecific antibody, was developed to function just like factor VIII does. As shown on the lower panel, emicizumab functions just like factor VIII, allowing factor IX to activate factor X, thus facilitating clot formation. This medication was recently FDA approved for use in patients with hemophilia A with or without factor VIII inhibitors. Thus more and more patients with hemophilia A are being treated with this medication. It is important to know because emicizumab interferes with the one-stage coagulation factor VIII assay.

Recall that the one-stage assay is based on the activated partial thromboplastin time test (APTT). The relationship between the APTT clotting time and estimation of the clotting factor VIII activity assay is such that the longer the APTT, the lower the measured factor VIII. Conversely the shorter the APTT, the higher the measured factor VIII will be. In the experiment shown in this slide, factor VIII-deficient plasma was spiked with progressively increasing concentrations of emicizumab. Although I have not shown the data, the APTT progressively shortens, and this results in an artifactual increase of the measured factor VIII level. In addition, measurement of factor VIII activity using a chromogenic assay with human reagents, as shown in the red color, will also overestimate the true factor VIII activity in the test plasma. However, as shown in green, when using a chromogenic assay with bovine reagents, one does not see this false elevation of factor VIII activity. In conclusion, the one-stage factor VIII activity assay and the chromogenic factor VIII activity assay using human substrate result in an artifactual elevation of factor VIII activity in the presence of emicizumab.

Implications for Factor VIII Inhibitor Bethesda Assay

The implications of these findings are important for detection of factor VIII inhibitors using the Bethesda assay. Using a one-stage factor VIII assay and chromogenic factor VIII assay with human reagents will result in a false negative or lower estimates of true Bethesda titers. For patients on emicizumab, it is very important to ensure that the laboratory performing factor VIII inhibitor titers in patients on emicizumab is using bovine substrates in its chromogenic factor VIII assays.


In conclusion, options for monitoring new modified extended half-life recombinant factor concentrates may require one-stage assays with specific contact activators. Alternatively chromogenic assays would provide more accurate post-infusion test results. Selected patients with mild hemophilia A may be missed with either the one-stage or the chromogenic assay, but more commonly, the disease severity may be misclassified. In this situation, if a male patient with a clear bleeding disorder has a normal one-stage factor VIII activity assay, one should consider follow-up testing with the chromogenic factor VIII activity assay. Finally, Bethesda titer assessment in patients on emicizumab should always have the testing performed using chromogenic factor VIII assay based on bovine regents.

Thank you for your attention.


  1. Peyvandi F, Oldenburg J, Friedman KD: A critical appraisal of one-stage and chromogenic assays of factor VIII activity. J Thromb Haemost. Feb 2016;14(2):248-261.
  2. Trossaert M, Lienhard A, Nougier C, et al: Diagnosis and management challenges in patients with mild haemophilia A and discrepant FVIII measurements. Haemophilia. Jul 2014;20(4):550-558.
  3. Pavlova A, Delev D, Pezeshkpoor B, et al: Haemophilia A mutations in patients with non-severe phenotype associated with a discrepancy between one-stage and chromogenic factor VIII activity assays. Thromb Haemost. May 2014;111(5)851-861.
  4. Kihlberg K, Strandberg K, Rosén S, et al: Discrepancies between the one-stage clotting assay and the chromogenic assay in haemophilia B. Haemophilia. Jul 2017;23(4)620-627.
  5. Oldenburg J, Mahlangu J, Kim B, et al: Emicizumab Prophylaxis in Hemophilia A with Inhibitors. N Engl J Med. 207;377:809-818.
  6. Calatzis A, Chen D, Adamkewicz J. Emicizumab: effects and interference on coagulation assays. ISLH 2017 Abstract Proceedings. Int J Lab Hematol 2017; 39(S2):24.

MCL Education

This post was developed by our Education and Technical Publications Team.