Article published online: 2022-12-06

THIEME
609

Factor VIII and Factor IX Activity Measurements

for Hemophilia Diagnosis and Related
Treatments
Annette E. Bowyer, FIBMS, PhD1 Robert C. Gosselin, CLS2

1 Department of Coagulation, Royal Hallamshire Hospital, Sheffield, Address for correspondence Robert C. Gosselin, CLS, Hemostasis and
United Kingdom Thrombosis Center, University of California, Davis Health System,
2 Hemostasis and Thrombosis Center, University of California, Davis Sacramento, CA 95817 (e-mail: [email protected]).
Health System, Sacramento, California

Semin Thromb Hemost 2023;49:609–620.

Abstract Accurate measurement of clotting factors VIII (FVIII) or IX (FIX) is vital for comprehen-
sive diagnosis and management of patients with hemophilia A or B. The one-stage
activated partial thromboplastin time (aPTT)-based clotting assay is the most com-
monly used method worldwide for testing FVIII or FIX activities. Alternatively, FVIII and
FIX chromogenic substrate assays, which assess the activation of factor X, are available
in some specialized laboratories. The choice of reagent or methodology can strongly
influence the resulting activity. Variation between one-stage FVIII or FIX activities has
been reported in the measurement of some standard and extended half-life factor
Keywords replacement therapies and gene therapy for hemophilia B using different aPTT
► one-stage factor reagents. Discrepancy between one-stage and chromogenic reagents has been
assays demonstrated in some patients with mild hemophilia A or B, the measurement of
► chromogenic factor some standard and extended half-life factor replacement therapies, and the transgene
assays expression of hemophilia A and B patients who have received gene therapy. Finally, the
► factor VIII measurement of bispecific antibody therapy in patients with hemophilia A has
► factor IX highlighted differences between chromogenic assays. It is imperative that hemostasis
► hemophilia laboratories evaluate how suitable their routine assays are for the accurate measure-
► gene therapy ment of the various hemophilia treatment therapies.

The X-linked, hemostatic disorders of hemophilia are caused IU/dL), and mild (FVIII:C or FIX:C > 5 to <40 IU/dL) disorders
by the absence or reduction of clotting factor VIII (hemo- based on the level of clotting factor activity.2 Patients with
philia A, HA) or factor IX (hemophilia B, HB), which can lead mild HA or HB have fewer bleeding problems than those with
to uncontrolled bleeding. It is estimated that more than moderate or severe forms, often only requiring replacement
300,000 people have hemophilia worldwide.1 The lower factor therapy following significant trauma or postopera-
the measurable factor VIII (FVIII:C) or factor IX (FIX:C) tively. Patients with moderate hemophilia may bleed follow-
functional “coagulant” activity, the more significant the ing minor trauma, whereas severely affected patients may
bleeding diathesis. HA and HB are classified into severe exhibit spontaneous bleeding, which can occur into joint
(FVIII:C or FIX:C < 1 dL), moderate (FVIII:C or FIX:C 1–5 spaces (hemarthroses). Bleeding in untreated severe

article published online Issue Theme Laboratory © 2022. The Author(s).

December 6, 2022 Diagnostics for Thrombosis and This is an open access article published by Thieme under the terms of the
Hemostasis Testing—Part II; Creative Commons Attribution-NonDerivative-NonCommercial-License,
Guest Editors: Robert C. Gosselin, permitting copying and reproduction so long as the original work is given
CLS and Kristi J. Smock, MD appropriate credit. Contents may not be used for commercial purposes, or
adapted, remixed, transformed or built upon. (https://creativecommons.org/
DOI https://doi.org/ licenses/by-nc-nd/4.0/)
10.1055/s-0042-1758870. Thieme Medical Publishers, Inc., 333 Seventh Avenue, 18th Floor,
ISSN 0094-6176. New York, NY 10001, USA
610 Factor Assays in Hemophilia Bowyer, Gosselin

hemophilia patients is variable, with annualized bleed rates or chromogenic substrate assays [CSA]).17,18 Since that time,
(ABR) ranging from 0 to more than 50.3 Treatment of hemo- there has been a proliferation of new hemophilia treatment
philia may be episodic (on-demand) following a bleed, or strategies, including modified (polyethylene glycol(PEG)
prophylactic (to prevent future bleeds) via regular injections ylated, albumin-fused, FC-fusion) extended half-life (EHL)
of factor concentrate. Prior to the last decade, the treatment replacement products or gene therapy (in lieu of factor
of hemophilia was with standard half-life (SHL) plasma- replacement). Each of these has laboratory challenges in
derived (pd) or recombinant (r) FVIII or (r)FIX. More recently, accurately measuring factor activity.19,20
modifications to rFVIII or rFIX molecules have extended the
half-life of the products in the circulation by around 1.5-fold
FVIII and FIX Factor Assays: General
for FVIII4–7 and up to 5-fold for FIX,8–10 while novel rebalanc-
Laboratory Considerations
ing therapies and gene therapy have greatly expanded the
treatment options for hemophilia.11–13 The prophylactic Unless the equipment and related reagents are designated for
dosage of clotting factor concentrates may be regulated by a hemophilia treatment center, it is likely that instrumenta-
a specific regimen of standard doses or tailored to each tion and related reagent selection for clotting factor assays
individual patient following measurement of the peak level will be predicated on modified PT and aPTT assay testing.
(the FVIII:C or FIX:C immediately after treatment) and With the understanding that FVIII and FIX testing may be
trough level (the lowest FVIII:C or FIX:C immediately prior used outside the scope of hemophilia assessment, there are
to the next dose). The hemostasis laboratory serves a critical certain expectations that should be considered when using
role in the diagnosis and management of HA or HB by these platforms outside the general-purpose use of PT/aPTT
providing screening tests (prothrombin time [PT] and acti- screening or drug-monitoring testing, including but not
vated partial thromboplastin time [aPTT]), specific factor limited to the following:
assays (e.g., factor VIII, F8), and/or inhibitor studies (e.g.,
Bethesda assay). Factor assays can be used for diagnostic • Variables associated with instrumentation, calibrator
purposes (e.g., identifying a congenital or acquired factor source, calibration type and LLOQ, aPTT reagent, factor-
deficiency), monitoring purposes (assessing the pharmaco- deficient plasma source, and sample diluent (►Table 1).
kinetics of a factor replacement therapy), assessing product Likely, many of these variables are default protocols
quality control (e.g., FVIII:C in cryoprecipitate), or to assess embedded within an instrument testing menu. Modifica-
product potency of factor concentrates. tions or alterations of these defaulted protocol(s), includ-
Historically, the problems associated with these assays in ing alternative reagents or calibrators, may constitute an
the diagnosis and management of hemophilia have been in-house or laboratory-developed test which may have
attributed to the variability of results between assays, regional regulatory requirements for validation prior to
usually secondary to test methodology, calibration, and clinical use.
reagent (including factor deficient material) sources.14,15 • The selection of whether to use OSA or CSA may be
Improvements in factor assay performance within and be- predicated on additional considerations or restrictions
tween laboratories have emerged with advancing technolo- such as reagent contracts, instrumentation, accreditation,
gies (automated analyzers), laboratory performance or regional regulatory requirements.
guidelines such as those from the British Committee for • Anticoagulant interferences including heparins, parenter-
Standardization in Haematology [BCSH], and proficiency al direct thrombin inhibitors (DTIs), and direct oral anti-
testing.16 coagulants (DOACs) may interfere with accurate
While biases still exist between laboratories in FVIII and performance of either OSA or CSA methods, with possible
FIX performance, in-house performance of these tests is underestimation of factor activity. Some CSA assays may
usually constrained by operational limitations. These are be less affected than other assays due to heparin neutral-
typically instrument related, such as differences in the lower izers in reagents and higher sample dilutions. Some drugs
limit of quantitation (LLOQ) and can impact on determining may mimic a factor inhibitor and exhibit assay non-
hemophilia severity. parallelism.21
Clinicians are often unaware of laboratory limitations, • Nonspecific inhibitors (e.g., lupus anticoagulant) or other
and most have a relatively naive knowledge of their labora- drug effects (e.g., lipoglycopeptide antibiotics) may inter-
tory performance in clotting factor or inhibitor assays. fere with OSA testing, although the inhibitor effect may be
Replacement products for treating HA and HB that were diminished due to sample dilutions for OSA testing.22–24
human (sometimes porcine) derived provided clotting factor CSA assays may be less affected due to higher sample
activities as expected when using traditional laboratory dilution.
methods. The expectation that all replacement therapies • Porcine-derived products (Obizur, porcine rFVIII,
could be reliably monitored by any reagent or methodology BAX801) are reliably assessed using OSA methods; CSA
changed when a B-domain–deleted (BDD) rFVIII (ReFacto, methods are less reliable.25–28
Wyeth Pharmaceutical) was approved by the U.S. Food and • For animal samples, OSA may be the preferred (only)
Drug Administration (FDA) in 2000. It was demonstrated that option, although animal factor levels may not be compa-
a ReFacto-specific calibrator was required to obtain accurate rable to humans, and thus calibration alterations may be
results, regardless of method (one-stage clotting assay [OSA] required.29

Seminars in Thrombosis & Hemostasis Vol. 49 No. 6/2023 © 2022. The Author(s).
 Factor Assays in Hemophilia Bowyer, Gosselin 611

Table 1 General factor assay considerations

Automation Most coagulation analyzers have the capacity to perform OSA factor assays, usually dedicated to a single
source of factor-deficient plasma, calibrator, and controls with clot detection by optical or mechanical
means. Few instruments are programmed for CSA factor methods. Alterations to the instrument protocol
may be required when using alternative materials and may require additional validation as required by
regional regulatory agencies. Analyzers may have a dual OSA platform to accurately assess low factor
activity levels (e.g., <15 IU/dL). Instruments without CSA protocols would require programming to adapt
the methodology into an automated platform. CSA methods may also require dual platforms, where the low
factor activity measurements requiring longer incubation and read times. Automation provides automatic
calibration services, calibration curve fitting, patient calculations, and some instruments provide
parallelism check to indicate presence of inhibitor
Calibration All OSA and CSA methods require a calibration to provide a quantitative measurement. Calibrators should be
traceable to recognized standard from reputable organizations such as World Health Organization (WHO),
National Institute for Biological Standards and Control (NIBSC), or International Society of Thrombosis and
Haemostasis (ISTH), and have an assigned activity using either OSA or CSA method. OSA methods typically
have 5–7 calibration points, whereas CSA methods may have less, but more than 2 calibration points.
Calibration curves can be linear, log–log; linear–log, polynomial, and use of derivatives. Patient samples
should be tested at least at three different dilutions for OSA, and single dilution for CSA method
Factor-deficient Factor-deficient plasma (DP) can be immunodepleted, chemical depleted, or from congenital deficiency
plasma sources (with or without VWF in FVIII DP) and must contain <1 IU/dL of factor. DP may be lyophilized or
frozen plasma. Whether one source is better than the other is for debate, each with their own advantages
and disadvantages (e.g., cost, stability, reuse or freezing, instrument compatible without transferring
to secondary vials, VWF levels, etc.)
aPTT reagent Most commercial aPTT reagents are designed to be sensitive to changes in FVIII and FIX levels. Activator
content (silica, kaolin, ellagic acid, polyphenols) and phospholipid type and source (e.g., animal, plant, or
synthetic) are confounders to responsiveness of factor testing. Reagent considerations are required when
the EHL factor testing is being performed on patients treated with EHL replacement products. Note that use
of OSA for QC purposes, especially on cryoprecipitate products, may require a higher predilution of the
sample than required with patient samples
Diluent Suitable diluents used for making calibrator and plasma sample diluents include saline or buffered solutions
such as Owren’s, Owren-Koller, HEPES, imidazole buffer, FVIII or FIX-deficient plasma, and others

Abbreviations: aPTT, activated partial thromboplastin time; CSA, chromogenic assay; EHL, extended half-life; HEPES, 4-(2-hydroxyethyl)-1-piperazine
ethane sulfonic acid; OSA, one-stage assay; VWF, von Willebrand factor.

FVIII and FIX Measurements for Diagnosis phatidylcholine, phosphatidylinositol, phosphatidylethanol-

amine, or sphingomyelin.35 The sensitivity of aPTT reagents
The separation of hemophilia into HA and HB was first made to mild deficiencies of FVIII and FIX is inconsistent.36–38
in 195230 following the development of the OSA and two- While it is recommended that a prolongation to the aPTT
stage clotting assays.31–33 A variation of the OSA has been in be present when FVIII, FIX, or FXI are less than 30 IU/dL,39 it
world-wide use since that time, whereas the two-stage follows that some reagents may have a normal aPTT in the
clotting assay is now performed only in a handful of special- presence of mild HA or HB which may compromise diagnosis.
ized laboratories. The OSA is a modification to the aPTT by In the 1980s, a chromogenic substrate FVIII assay (CSA)
dilution of test plasma and addition of plasma that is was introduced.40 This assay involves activation of the test
completely devoid of the clotting factor to be tested (i.e., FVIII, then, in combination with added FIXa, activates FX to
either FVIII or FIX) for hemophilia diagnosis. This factor- FXa. The FXa generated cleaves a FXa-specific chromophore
deficient plasma has a prolonged aPTT and any FVIII or FIX and the resultant change in color is measured via optical
present in the patient plasma will shorten (or “correct”) the density (OD). The OD of the test plasma is compared with
aPTT. The clotting times generated in the patient plasma are those of a reference or calibrator plasma and the concentra-
compared with those of a reference or calibrator plasma tion determined via a calibration curve. The higher the color
and the concentration determined via a calibration curve.34 generation, the greater the level of FVIII. There are several
The OSA is a heterogeneous test, with numerous combina- kits on the market, which vary in source of reagents (human,
tions of reagents and instrumentation used worldwide. bovine, or a mix), phospholipid type and concentration,
General considerations for factor assays include the use of buffers, and incubation time.
automation, calibration, factor-deficient plasma source, aPTT The chromogenic FIX assay is a recent addition to the
reagent source, number of plasma dilutions, and diluent laboratory repertoire. The assay principle is also a generation
(►Table 1). aPTT reagents contain a contact activator, with of FXa, then measurement by cleavage of a specific chromo-
a variety of materials used, including ellagic acid, kaolin or genic substrate. There are, at the time of writing, three
silica derivatives, as well as a variety of animal or plant- chromogenic FIX assay kits available which vary in their
sourced phospholipids including phosphatidylserine, phos- constituents and assay conditions.41–44

Seminars in Thrombosis & Hemostasis Vol. 49 No. 6/2023 © 2022. The Author(s).
612 Factor Assays in Hemophilia Bowyer, Gosselin

FVIII and FIX Measurements: Discrepancies Assay discrepancy in HB is rarely described, but when
in Mild Hemophilia described appears to compromise the severity with patients
alternating between moderate and mild HB. One report
The diagnosis of severe, moderate, and some mild HA and HB described 14 of 15 patients with FIX: c.572G > A; p.Arg191His
phenotypes can be made by either OSA or CSA. The results of or FIX: c.571C > T; p.Arg191Cys amino acid changes in F9. The
the two methods are generally comparable. However, there are mean OSA was 0.02 IU/mL and that of CSA was 0.06 IU/mL and
more than 20 mutations in F8 which are linked to significant patients reported a mild bleeding phenotype.60 A further study
discrepancy between OSA and CSA or two-stage clotting reported 2.5-fold higher results by OSA compared with CSA.61
assay.31 Assay discrepancy was initially reported in 1983 in The aPTT reagent composition has also been demonstrated to
four patients from two families with a twofold or greater FVIII: affect the OSA in patients with mild HB.62
C measured by OSA than two-stage clotting assay.45 This lower
two-stage clotting or CSA form of assay discrepancy has been
FVIII and FIX Measurements: Monitoring of
widely reported in Europe and Australia46–50 and is caused by
Standard Half-Life FVIII and FIX
point mutations in F8 at the A1–A2, A2–A3, or A1–A3 domain
interfaces. These have been reported to increase the rate of The monitoring of pdFVIII or FIX and most SHL recombinant
dissociation of A2 domain leading to premature inactivation of products are not influenced by OSA reagents. The exception
FVIII.51–53 The CSA has a longer incubation time than OSA; so, is the B-domain–depleted (BDD) rFVIII concentrate, ReFacto,
the untimely inactivation of FVIII manifests as reduced CSA and its successor ReFacto AF (moroctocog alfa, Pfizer63).
activity, whereas the OSA FVIII:C is less impacted. The reverse FVIII:C levels of ReFacto AF measured by OSA calibrated
discrepancy with a lower OSA compared with CSA has also with a reference plasma may be 20 to 50% lower than
been described. A further 10 mutations in F8 have been linked expected; however, this difference can be reduced by use
to this type of assay discrepancy but in particular one muta- of either a product-specific reference plasma (ReFacto labo-
tion, p.Tyr365Cys/Phe has been commonly described in the ratory standard, Pfizer) in a standard OSA or using a plasma
United Kingdom.54–57 These mutations, along with p. reference plasma in a CSA.18 The recovery of ReFacto AF may
Glu340Lys and p.Ile388Thr, are thought to induce a conforma- also depend on the constituents of the aPTT reagent since not
tional change resulting in a delay to thrombin activation of all aPTT reagents demonstrate lower results by OSA.64,65
FVIII at p.Arg391.54,58,59 This causes a lower OSA since FVIII is Chromogenic FVIII assays also recover close to the expected
not activated as quickly as wild-type FVIII, but the longer FVIII:C with SHL products; however, some SHL rFIX products
incubation time in the CSA results in higher or normal activity. may be underestimated by chromogenic FIX assays.66,67 The
The reasons for reverse discrepancy with other mutations that United Kingdom Haemophilia Centre Doctors’ Organization
are not near this thrombin activation site are unclear.50 Some (UKHDCO) has published guidance for the laboratory moni-
HA patients with FVIII assay discrepancy have reduced, but still toring of FVIII and FIX concentrates.68 A summary of poten-
discordant, OSA and CSA; so, the diagnosis is not usually tial differences can be viewed in ►Table 2.
compromised. However, in those patients where one result
is within normal limits and the second is reduced, there may be
FVIII and FIX Measurements: Monitoring
a delay to diagnosis if only one assay method is routinely
Extended Half-Life FVIII and FIX
performed. It is therefore important to measure both OSA and
CSA in patients with suspected mild HA. The clinical bleeding One of the disadvantages of treatment with SHL FVIII and FIX
phenotype generally coincides with the lowest value observed, therapy is the length of time that the product remains active
but this is dependent on the mutation present.53 in the circulation; for FVIII, this is approximately 12 hours

Table 2 Reagent and methodological discrepancy in the measurement of FVIII and FIX molecules

Molecule One-stage V chromogenic One-stage assay Chromogenic assay References

assay discrepancy reported reagent differences reagent differences
48,56
Native FVIII Yes in some patients with mild HA No No
125
Pd FVIII Yes No No
65,118,126
SHL FVIII Yes with BDD Yes with BDD No
68,81,84,127
EHL FVIII Yes Yes No
100,102,128
FVIII mimetics Yes Not suitable for use Yes
60,62,129
Native FIX Yes in some patients with mild HB No No data
66
Pd FIX No Yes but limited data No
66,90,130
SHL FIX Yes Yes Yes
66,79,90
EHL FIX Yes Yes Yes

Abbreviations: BDD, B-domain deleted; EHL, extended half-life; HA, hemophilia A; HB, hemophilia B; Pd, plasma derived; SHL, standard half-life.

Seminars in Thrombosis & Hemostasis Vol. 49 No. 6/2023 © 2022. The Author(s).
 Factor Assays in Hemophilia Bowyer, Gosselin 613

Table 3 Modified factor VIII replacement products131–135

Name Manufacturer Factor VIII modification Half-lifea (h) Approval date

ELOCTATE/ELOCTA Bioverativ/SOBI Fusion to Fc domain of IgG1 13–20 FDA Jun 2014
(rFVIII-Fc BDD) EMA Nov 2015
AFSTYLA CSL Behring Single chain—PEGylated 10–14 FDA May 2016
(CSL627) EMA Nov 2015
ADYNOVATE/ADYNOVI Takeda (Shire) 20-kDa branched PEGylated 12–15 FDA Dec 2016
(Bax 855) EMA Jan 2018
JIVI Bayer Site-specific 60-kDa PEGylated 17–21 FDA Aug 2018
(BAY 94–9027) EMA Nov 2018
ESPEROCT Novo Nordisk 40-kDa glycoPEGylated 10–14 FDA Feb 2019
(N8-GP) EMA Apr 2019

Abbreviations: EMA, European Medicines Agency; FDA, Food and Drug Administration; KDa, kilodalton; PEG, polyethylene glycol.
a
Half-life represents mean values, and is age dependent (pediatrics vs. adults)—source is prescribing information from each respective drug.

and for FIX approximately 20 hours.69 There have been Nordisk, Denmark) was underestimated by up to 50%
several approaches undertaken to extend the half-life of by silica-activated aPTT SP,80,81 while Jivi (previously Bay
each protein including fusion with polyethylene glycol 94–9027-, Bayer AG, Germany) was underestimated by some
(PEG),5,6,70–72 albumin,73,74 or the Fc fragment of IgG.75–77 silica-based aPTT reagents and overestimated by some
This has resulted in commercially available EHL FVIII kaolin-based aPTT reagents.82 The single-chain recombinant
(►Table 3) and FIX (►Table 4) products for the treatment FVIII, Afstyla (CSL Behring, Germany), is underestimated by
of HA and HB, respectively. approximately 45% by OSA,83,84 and consequently the man-
Even with liberal acceptability for factor recovery of 25 to ufacturer recommends the CSA.85 The manufacturer also
30%, it became apparent during clinical and laboratory field states that if the OSA is used, then a correction factor of 2 can
trials with some EHL products and some aPTT reagents that be applied to obtain the final result. This approach is not
significant discrepancy in measured FVIII or FIX recovery internationally recommended due to differences between
could be reproducibly demonstrated. It cannot be assumed one-stage assays.68 The CSA has been reported to achieve
that EHL molecules that use the same modification technol- acceptable recovery with all currently licensed EHL FVIII
ogy (such as PEG) will exert the same effect on aPTT reagents concentrates. Additional modifications to rFVIII, which
that share the same activator and phospholipid source. combines BDD rFVIII with Xten polypeptides and a fragment
Likewise, it cannot be assumed that aPTT reagents that of von Willebrand factor, have improved the half-life exten-
share the same activator and phospholipid source will sion to more than threefold compared with SHL in clinical
have the same response to those EHL with the same modifi- studies.86
cation.78,79 EHL prescribing information may assist clinicians There are currently three EHL FIX concentrates licensed
for acceptable or recommended laboratory methods, but it is for use in some countries. These have been modified by Fc
unlikely that many clinicians are aware of the FVIII or FIX fusion,77 albumin fusion,9 or glycopegylation87 (►Table 4).
method used in their laboratory and the limitations of those Under or over estimation with aPTT reagents in the OSA has
assays. Additionally, most physicians are usually unaware of been reported for each product.66,79,88–90 CSAs are also
their laboratory’s performance in FVIII, FIX, or related inhib- varied in their response.66,91
itor assays as assessed by External Quality Assurance pro- In 2020, there were three guidance publications that
grams required for comparing local laboratory performance provided recommendations for or against FVIII and FIX
to regional or international peers. OSA or CS assays for EHL products.68,92,93 Of particular
There are currently five FVIII EHL products available for concern is (1) limited data for nondescribed aPTT reagents,
clinical use (►Table 3). Esperoct (previously N8-GP, Novo (2) insufficient or discordant data obtained from clinical or

Table 4 Modified factor IX replacement products136–138

Name Manufacturer Factor VIII modification Half-lifea (h) Approval date

ALPROLIX Bioverativ/SOBI Fusion to Fc domain of IgG1 68–94 FDA Mar 2014
(rFIX-Fc) EMA May 2016
IDELVION CSL Behring Fusion to albumin
90 FDA Mar 2016
(rIX-FS CSL654) EMA May 2016
REBINYN/REFIXIA (N9 ¼ GP) Novo Nordisk 40-kDa glycoPEGylated 70–89 FDA May 2017
EMA Mar 2017

Abbreviations: EMA, European Medicines Agency; FDA, Food and Drug Administration; kDa, kilodalton; PEG, polyethylene glycol.
a
Single-dose, half-life mean values, and is age dependent (pediatrics vs. adults)—source is prescribing information from each respective drug.

Seminars in Thrombosis & Hemostasis Vol. 49 No. 6/2023 © 2022. The Author(s).
614 Factor Assays in Hemophilia Bowyer, Gosselin

Fig. 1 Recommended or rejected OSA methods for measuring FVIII and FIX EHL products from recent publications. 68,92,93 Columns labeled “1”
reflect recommendations from Peyvandi et al, 92 columns labeled “2” from Gray et al, 68 and columns labeled “3” from Jeanpierre et al.93 Green
cells indicate a recommended method, yellow cells indicate insufficient or conflicting clinical or field trial data, and red cells indicate rejected
method. A white cell indicates this method was not specifically addressed by authors. Note: Afstyla can only be measured using a suitable FVIII
chromogenic assay. Note: For Adynovate/Adynovi, the Kihlberg et al 60 group could not recommend any OSA method. PL, phospholipid; RBT,
rabbit brain thromboplastin.

field studies, and (3) discordant OSA or CSA recommenda- not adequately described or recommended, implementing a
tions between these publications. It is likely that a clinical reagent for EHL based on other reagent platforms with
laboratory will provide a single OSA method for both FVIII similar activator may not be a suitable means for predicting
and FIX, usually due to contractual agreements or instru- suitability for EHL monitoring (►Fig. 3). Given the variability
ment default methods. Therefore, when evaluating the three within a given reagent platform (►Figs. 1 and 2), it is likely
guidance documents, it is necessary to recommend which that more than one FVIII or FIX method may be required to
OSA method is suitable for monitoring FVIII and FIX EHL accurately monitor all EHL replacement therapies.
concentrates (►Fig. 1). For CSA methods, there was mostly
concordance between these guidance recommendations,
FVIII Measurements: FVIII Mimetics
with notable exception for Esperoct and Idelvion (►Fig. 2).
It is imperative that laboratories assess their local assays FVIII mimetics, including emicizumab (Hemlibra, Roche
for suitability to measure EHL FVIII and FIX concentrates that Chugai) and Mim8 (Novo Nordisk), are humanized bispecific
may be used to treat their patients. For those aPTT methods antibodies directed to human FIX (FIXa) and FX, which

Fig. 2 Recommended or rejected CS methods for measuring FVIII and FIX EHL products from recent publications. 68,92,93 Columns labeled “1”
reflect recommendations from Peyvandi et al, 92 columns labeled “2” from Gray et al, 68 and columns labeled “3” from Jeanpierre et al.93 Green
cells indicate a recommended method, yellow cells indicate insufficient or conflicting clinical or field trial data, and red cells indicate rejected
method. A white cell indicates this method was not specifically addressed by authors. § Generic recommendations for CS VIII methods, no specific
reagent(s) may be noted. Note: Kihlberg et al 60 group did not provide recommendations for Adynovate/Adynovi based on limited clinical or field
trial data.

Seminars in Thrombosis & Hemostasis Vol. 49 No. 6/2023 © 2022. The Author(s).
 Factor Assays in Hemophilia Bowyer, Gosselin 615

Fig. 3 Aligning aPTT reagents by activators (silica or polyphenols) of the recommended or rejected CS methods for measuring FVIII and FIX EHL
products from recent publications. 68,92,93 Columns labeled “1” reflect recommendations from Peyvandi et al, 92 columns labeled “2” from Gray
et al,68 and columns labeled “3” from Jeanpierre et al. 93 Green cells indicate a recommended method, yellow cells indicate insufficient or
conflicting clinical or field trial data, and red cells indicate rejected method. A white cell indicates this method was not specifically addressed by
authors. PL, phospholipid; RBT, rabbit brain thromboplastin.

activate FX in the absence of FVIIIa.94,95 Emicizumab is FVIII and FIX Measurements: Gene Therapy
licensed for use in Europe, Australia, and the United States,
in patients with HA and anti-FVIII antibodies and severe HA Gene therapy trials have been running for FVIII and FIX for
without antibodies.96 Bispecific antibodies do not require several years, with numerous currently active and recruiting
preactivation to be functional, unlike FVIII; thus, the action clinical trials for both HA and HB.12,110–112 Recent guidance
on FX is more rapid97 and this impacts on some hemostasis from the U.S. FDA for human gene therapy in hemophilia has
tests.98–100 The aPTT dramatically shortens, even at subther- a section detailing the necessity for laboratory testing to
apeutic concentrations and any aPTT-based assays will also include both chromogenic and one-stage assays with a
be affected, including OSA for FVIII.98,101 OSA FVIII is artifi- variety of reagents in vitro and a comparative field study
cially increased, often far above the top of the reference in patient plasma.113
range. Several organizations have published guidance for the Differences in FVIII or FIX transgene expression measured
monitoring of patients receiving emicizumab therapy.102,103 by OSA and CSA have been reported in hemophilia patients
Quantitative measurement of the emicizumab drug concen- who have received certain gene therapy products (►Table 5).
tration can be made by modifying the OSA to use an In HA, an approximate 1.6-fold higher FVIII:C OSA FVIII:C than
emicizumab-specific calibrator and high plasma dilutions. CSA has been reported.114–117 Curiously, this is the reverse of
The use of CSA which use bovine FX can measure endogenous the pattern observed in some BDD-rFVIII molecules.17,118
FVIII due to their insensitivity to emicizumab at therapeutic Rosen et al proposed that the higher OSA compared with
concentrations.104 The monitoring for FVIII inhibitors in CSA in AAV5-FVIII-SQ molecules is caused by accelerated early
patients receiving these products create an additional chal- FXa and thrombin generation, and shorter clotting times
lenge, with modification to existing Bethesda or Nijmegen generate higher reported FVIII activity levels. This trend was
methods having been described.105 recorded in transgene FVIII:C and the rFVIII-SQ molecule.119
Kinetic assays highlighted the assay differences, but the
underlying mechanism is currently unknown.
FVIII and FIX Measurements: Rebalancing
There has been a difference in recovery of FVIII:C with a
Therapies
limited number of different aPTT reagents in the clinical
New classes of drugs, not based on the replacement of FVIII trials. Ideally, laboratory field studies would provide perfor-
or IX molecules, are currently in clinical trials for prophy- mance characteristics for OSA or CS methods not used in the
laxis of hemophilia patients. They include the small inter- clinical trials.119
ference RNA (siRNA) antithrombin knockdown therapy Discrepancy between OSA and CSA FIX:C has also been
(fitusiran, Sanofi),106 anti–tissue factor pathway inhibitor reported following FIX gene therapy for HB. Gene therapy
(TFPI) monoclonal antibodies (concizumab, Novo Nordisk, approaches that use FIX-Padua, a naturally occurring muta-
and marstacimab, Pfizer),107,108 and serpins against acti- tion with higher FIX:C than wild-type FIX,120 have observed
vated protein C.109 These drugs are able to increase throm- differences in one-stage FIX:C measured using a variety of
bin generation in the absence of FVIII and FIX, although it is aPTT reagents and also between OSA and CSA in both the
unlikely that monitoring these rebalancing therapies using transgene FIX expressed by patients and plasma spiked with
routine hemostasis assays will be clinically useful, or easily the FIX molecule.121–124 The degree of OSA versus CSA
applied. discrepancy may be up to twofold; again, OSA activities

Seminars in Thrombosis & Hemostasis Vol. 49 No. 6/2023 © 2022. The Author(s).
616 Factor Assays in Hemophilia Bowyer, Gosselin

Table 5 Selected OSA methods from clinical trials for FVIII and FIX gene therapy

OSA/CA (slope) OSA/CA (ratio) OSA/CA (slope)

Hemophilia A119 Hemophilia B121 Hemophilia Ba
Actin FS 1.29–1.88 ND 2.03
Actin FSL 1.52–1.66 1.2–1.6 1.10
SynthasIL 1.53–2.01 1.3–2.4 1.38
Triniclot aPTT HS 1.67 ND ND
CK Prest ND 0.7–2.2 ND
PTT-Automate ND 1.0–2.8 ND
Pathromtin SL ND ND 1.49

Abbreviations: CA, chromogenic assay; ND, no data; OSA, one-stage clotting assay.
a
Author (RCG) unpublished data.

are higher than CSA, but the degree of discrepancy between Sobi, and personal fees from Pfizer, outside the submitted
OSA and CSA seems to vary between patients.121 work.
With noted observations of differences between OSA and R.C.G. reports personal fees from Sysmex America Incor-
CS methods in clinical studies, the primary endpoint in porated and from Diagnostica Grifols, and honoraria from
hemophilia gene therapy is the phenotype of the patient. Diagnostica Stago, outside the submitted work.
It is unclear whether routine monitoring is required for gene
therapy but is likely necessary for patients who require
surgical intervention. It is unknown whether additional References
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