Performance Evaluation of Different FIX Activity Assays for Determining Nonacog Beta Pegol (N9-GP, Refixia®) Activity Using Reagent-/Platform-Specific Conversion Factors

Abstract

Background

Nonacog beta pegol (N9-GP) is a glycoPEGylated FIX replacement product with extended half-life for treatment of haemophilia B patients. Monitoring of N9-GP with clotting-based one-stage FIX assays is complicated by high variations, mainly due to reagent-specific interference with polyethylene glycol.

Methods

In 11 distinct specialized coagulation laboratories in Austria, N9-GP spiked samples were measured in replicates in two distinct surveys, 3 years apart, using five different one-stage assay reagents and one chromogenic FIX assay. Regression analysis was used to investigate if back-calculation of N9-GP levels is feasible.

Results

We could demonstrate a linear relationship between the spiked N9-GP concentration and measured FIX activity levels for all examined assays, suggesting that N9-GP activity may be back-calculated using reagent-/platform-specific conversion factors. Within-laboratory variation after 3 years was acceptable in most, but not all, laboratories.

Conclusion

We demonstrate that back-calculation of N9-GP activity levels may be possible when using one-stage FIX assays. However, we recommend that every laboratory ascertain its own conversion factor. When measuring real patient samples, we encourage simultaneous measurement of N9-GP spiked control material with known concentrations to ensure the validity of the current back-calculation.

 

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Keywords

coagulation factor IX - haemophilia B - nonacog beta pegol - Refixia - aPTT

Summary

Nonacog beta pegol (N9-GP) is a new FIX preparation with prolonged half-life. The different compositions of aPTT reagents used in FIX assays markedly influence N9-GP activity measurements, including significant assay-dependent over- or underestimation. In the present multi-centre study, samples spiked with different N9-GP concentrations were measured in 11 specialized coagulation laboratories in Austria, using five different aPTT-based FIX activity assays and one chromogenic FIX test. Samples were measured in replicates in two distinct surveys separated by 3 years. Regression showed a linear relationship between true N9-GP concentrations and measured FIX activity for all aPTT reagents. Thus, a reagent-/instrument-specific back-calculation of FIX activity results due to N9-GP activity seems feasible. The variation over 3 years is acceptable in most but not all laboratories. Therefore, when using back-calculation, every laboratory should determine its own conversion factor and measure N9-GP-spiked control samples alongside patient samples to ascertain the actual calibration and calculation.

 

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Zusammenfassung

Nonacog beta pegol (N9-GP) ist ein neuartiges FIX Präparat mit verlängerter Halbwertszeit, das zur Therapie von Hämophilie B Patienten eingesetzt wird. Die Einstufen-, aPTT-basierte FIX-Bestimmung wird durch N9-GP durch unterschiedliche Zusammensetzungen der aPTT-Reagenzien stark beeinflusst, was das Monitoring deutlich erschwert. In der vorliegenden Studie wurden N9-GP-gespikte Proben unterschiedlicher Konzentration in elf spezialisierten Gerinnungslabors in Österreich mittels fünf verschiedener aPTT-basierter FIX Aktivitätstests und einem chromogenen FIX Assay in Replikaten zu zwei unterschiedlichen Studien-Zeitpunkten im Abstand von drei Jahren gemessen. Wir konnten mittels Regressionsanalyse für alle untersuchten Reagenzien zeigen, dass ein linearer Zusammenhang zwischen der N9-GP Konzentration und der gemessen FIX Aktivität besteht, wodurch eine Reagens-/Geräte-spezifische Rückrechnung der gemessenen FIX Aktivitäten auf die N9-GP Aktivität möglich scheint. Die Variabilität der Messungen innerhalb von drei Jahren war für die meisten, aber nicht alle, Labore akzeptabel. Jedes Labor sollte seinen eigenen Konversionsfaktor erheben und wir empfehlen, dass bei jeder Rückrechnung von realen Patientenproben Kontrollen mit bekannter N9-GP-Aktivität mitgeführt werden, um die aktuelle Rückrechnung auf ihre Richtigkeit hin zu überprüfen.

 

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Introduction

Haemophilia B (HB) is a rare congenital X-linked recessive bleeding disorder, characterized by a deficiency of coagulation factor (F) IX. More than 2,100 FIX mutations were described related to HB.[1] The estimated prevalence of HB per 100,000 males is about 3.2 to 4.4. Approximately one-third of patients have a severe type.[2] The extent of bleeding in HB usually correlates with the residual endogenous FIX levels. Therefore, HB disease severity is defined based on FIX plasma levels: severe <1%, moderate 1 to 5%, and mild >5 to 40%.[3] Commonly, patients with severe HB develop spontaneous haemorrhages but a mild bleeding phenotype may be seen in 10 to 15% of them.[4] Severe disease usually presents in infancy, whereas late onset manifestations are rare. The initial sites of bleeding differ depending on the age of onset and include the central nervous system, sites of invasive interventions (e.g., venipuncture), skin, muscles, joints, and mucous membranes.[5] [6] [7] Current treatment regimens of HB are based on intravenous FIX substitution therapy, either symptomatically in case of bleeding episodes or as prophylaxis to prevent bleeding and joint destruction.[8] The WFH guidelines for the management of haemophilia recommend prophylaxis over episodic therapy whenever feasible, with the goal of an absolute prevention of haemorrhagic events.[9] Previous treatment strategies predominantly focused on using purified plasma-derived FIX (pdFIX) concentrates and recombinant FIX (rFIX). However, these conventional FIX products have a relatively short plasma half-life of 18 to 24 h and thus require frequent administration, typically bi-weekly, to maintain FIX levels at >0.01 IU per mL (>1%).[10] In an effort to prolong the duration between applications in order to improve the quality of life of these patients, new FIX formulations with extended half-life (EHL) were developed. Novel concepts of EHL include protein fusion of rFIX to immunoglobulin Fc fragments[11] or albumin,[12] or by the covalent attachment of a polyethylene glycol (PEG) molecule (glycoPEGylation) to the rFIX activation peptide.[13] Nonacog beta pegol (N9-GP, Refixia®, Rebinyn®; Novo Nordisk A/S, Bagsværd, Denmark) is an intravenous glycoPEGylated rFIX product with an EHL of 93 hours in humans[14] and preserved enzymatic properties.[15] Many clinical trials, such as the ‘PARADIGM’ phase 3 trial, demonstrated efficacy and tolerability of N9-GP for the prevention and treatment of bleeding in HB patients.[13] [16] [17] [18] [19] Notably, as an alternative to lifelong FIX replacement therapy, adeno-associated virus 5 vector-based gene therapy (Etranacogene Dezaparvovec) has become available in recent years. It has shown promising results in terms of reducing the annualized bleeding rate and demonstrating a favourable safety profile in a phase 3 study.[20]

FIX activity monitoring is crucial after administration to guide optimal patient management and to improve patient safety.[21] Usually, activated partial thromboplastin time (aPTT)-based one-stage clotting assays (OSAs) are used for this purpose. Nevertheless, FIX chromogenic substrate assays (CSAs), e.g., the BIOPHEN Factor IX (Hyphen BioMed, Neuville-Sur-Oise, France) or the Rox Factor IX (Rossix, Mölndal, Sweden), became available lately and are increasingly used in highly specialized coagulation laboratories.[22] Significant variability was found between OSA and CSA in HB patients,[23] although the underlying molecular mechanisms for those discrepancies have yet to be clarified. The administration of conventional (unmodified, non-mutated) pdFIX and rFIX agents can cause differences in the results of FIX, measured by either OSA or CSA.[9] In EHL FIX monitoring by OSA, reagent-dependent over- or underestimation of biological FIX activity was reported which was mainly related to differences in the composition of the aPTT reagents (different activators and phospholipids).[22] In the monitoring of N9-GP, isolated studies suggested a putative superiority of ellagic acid-based activators compared to silica- and kaolin-based activators. However, scientific evidence surrounding this new research field is still scarce and there is a notable data divergence, which reflects reagent- and/or instrument-dependent differences. An overview of different test systems and N9-GP recovery performance is provided in [Tables 1] and [2].[10] [21] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] According to current guidelines, every coagulation laboratory should verify the local reagent–instrument combination for appropriateness to monitor a given EHL product.[9]

Table 1 aPTT-based one-stage FIX assays evaluated for the monitoring of N9-GP

Reagent	Activator	PL source	Manufacturer	N9-GP recovery	References
Acceptable
APTTest	Diatomaceous earth	Cephalin	Wiener Laboratories (Rosario, Argentina)	Acceptable (on STA Compact and Sysmex CS)	Duboscq et al[31]
DG Synth	Ellagic acid	Rabbit brain cephalin	Grifols	Acceptable (except for very low samples)	Bowyer et al[10]
STA-Cephascreen	Polyphenolic component	Cephalin	Diagnostica Stago	Acceptable	Duboscq et al,[31] Holm et al,[33] Tiefenbacher et al[32]
Dapttin TC	Silica and sulfatide	Highly purified PLs	Technoclone	Acceptable	Holm et al[33]
Divergent reports
Actin FSL	Ellagic acid	Purified soy phosphatides/rabbit brain phosphatides	Siemens	Acceptable (on STA Compact, Sysmex CS and ACL TOP) Underestimation	Duboscq et al[31] Bowyer et al[10]
*SynthAFax	Ellagic acid	Synthetic	IL	Acceptable (except for very low samples: 0.03 IU/mL) Acceptable Overestimation (on STA Compact, Sysmex CS and ACL TOP)	Bowyer et al[10] Hermit et al,[35] Holm et al,[33] Sørensen et al[24] Duboscq et al[31]
Actin	Ellagic acid	Rabbit brain cephalin	Siemens	Underestimation (on CSB100i and BCS XP) Overestimation (on STA Compact, Sysmex CS and ACL TOP)	Bowyer et al[10] Duboscq et al[31]
*STA-C.K. Prest	Kaolin	NA	Diagnostica Stago	Overestimation (on STA Compact) Underestimation	Duboscq et al[31] Persson et al[26]
Overestimation
APTT SP	Silica	Synthetic	IL (Bedford, MA, USA)	No (strong overestimation)	Bowyer et al,[10] Hermit et al,[35] Duboscq et al[31]
*Pathromtin SL	Silica, silicon dioxide particles	Vegetable	Siemens	Strong overestimation	Bowyer et al,[10] Duboscq et al[31]
Triniclot aPTT S	Silica	NA	Diagnostica Stago	Strong overestimation (on STA Compact, Sysmex CS, ACL TOP)	Duboscq et al[31]
Underestimation
*Actin FS	Ellagic acid	Purified soy phosphatides	Siemens	Underestimation	Bowyer et al,[10] Hermit et al,[35] Persson et al[26]
*SynthASil	Silica, colloidal	Synthetic	IL	Underestimation	Bowyer et al,[10] Duboscq et al,[31] Persson et al[26]

Abbreviations: NA, not assessable; N9-GP, nonacog beta pegol; PL, phospholipids.

Note: *The five aPTT reagents used by the laboratories in the present study.

Table 2 Chromogenic substrate FIX assays evaluated for the monitoring of N9-GP

Reagent	Manufacturer	N9-GP recovery	Selected references
*Biophen FIX	Hyphen BioMed	Acceptable	Bowyer et al,[10] Holm et al,[33] Tiefenbacher et al,[32] Sørensen et al[24]
ROX FIX	Rossix	Acceptable	Bowyer et al,[10] Holm et al,[33] Tiefenbacher et al[32]

Abbreviation: N9-GP, nonacog beta pegol.

Note: *The chromogenic FIX assay used by laboratories in the present study.

The aim of the study was to perform a comprehensive investigation of N9-GP on different reagent–instrument combinations and to evaluate possible reagent/instrument-specific conversion equations. Herein, we present the data from two sequential multi-centre inter-laboratory surveys. The first survey was intended to show the possibilities of measuring different N9-GP concentrations spiked into FIX-deficient plasma (supplied by Novo Nordisk) using different aPTT reagents and analyzers. A statistic evaluation of over- and underestimated test results showed that N9-GP activity could be back-calculated from respective FIX activities. The second survey served as ‘proof of principle’. We hypothesized that there is a linear relationship between the FIX activity value provided by the respective assays and the true N9-GP concentration, thus enabling an assay-/instrument-dependent conversion by using regression analysis and individual conversion factors.

 

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Methods

Participating Centres, Coagulation Platforms, and FIX One-Stage Assays

The first survey took place in 2019, while the second was postponed to 2022 (instead of 2020) due to the coronavirus disease 2019 pandemic. Samples with different N9-GP concentrations spiked into FIX-deficient plasma were sent to 11 different coagulation laboratories in Austria, with the request to perform their routine FIX tests (if available OSA and CSA) for precision and accuracy evaluation. The following four aPTT reagents in combination with FIX-deficient plasma were used in clot-based OSA by the 11 different laboratories, respectively. Actin FS (Siemens) on Siemens Atellica COAG360 (Institute of Laboratory Medicine, Hospital Wels-Grieskirchen) or Sysmex CS-5100 (Central Medical Laboratories Feldkirch, Institute of Laboratory Medicine, Kepler-University-Hospital Linz; Institute of Laboratory Medicine and Microbiology Klagenfurt; and Department of Laboratory Medicine, Medical University of Vienna; the latter used Sysmex CS-2500 in the second survey); Pathromtin SL (Siemens) on Siemens BCS XP (Central Institute of Medical and Chemical Laboratory Diagnostics, University Hospital of Innsbruck), Siemens Atellica COAG360 (Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz), or Sysmex CS-5100 (Department of Laboratory Medicine, Paracelsus Medical University of Salzburg); STA C.K. Prest (Diagnostica Stago) on Stago STA Max (Clinical Institute of Laboratory Medicine, University Clinic St. Pölten), SynthASil (Instrumentation Laboratory [IL]) on IL ACL Top 500 (Klinikum Hietzing, Central Laboratory), and SynthAFax (IL) on IL ACL 350 (Department of Pediatrics and Adolescent Medicine, Medical University of Graz).

 

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FIX Chromogenic Substrate Assays

Out of the 11 centres 7 performed CSA, all using the Hyphen Biomed BIOPHEN FIX assay on the platforms mentioned previously.

 

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N9-GP Study Sample Preparation

In the first survey, N9-GP sample kits were prepared and distributed by Precision BioLogic, Inc. An inhibitor-free congenital FIX-deficient plasma from an individual with severe HB (purchased from HRF Inc., Raleigh, NC, USA) with no detectable FIX activity (<0.01 IU/mL), was spiked with a single lot of N9-GP (HS64Y51, supplied by Novo Nordisk), at nominal levels of 0.9, 0.6, 0.2, and 0.0 IU/mL, based on the potency of certificate of analysis assigned by OSA. The drug product was reconstituted in the supplied histidine solvent solution, according to the manufacturer's instructions. Then, using FIX-deficient congenital plasma, 10X stock solutions were prepared. The 10X stocks were further diluted 1 in 10 with the same congenital FIX-deficient plasma to target the desired final concentrations. The same FIX-deficient congenital plasma was also used as a neat to represent the blank sample (∼0 IU/mL) in the kit. All products were dispensed in 1 mL cryovials flash-frozen in liquid nitrogen and stored at below −70°C. Each laboratory got five sample sets (one for each measuring day).

In the second survey, the company Technoclone (Vienna, Austria) prepared and shipped the N9-GP samples. N9-GP drug substance was first diluted in solvent achieving a concentration of 456 IU/mL. In a second step, further dilution in congenital HB plasma was performed to achieve a concentration of 40 IU/mL. Using commercial coagulation FIX-deficient plasma (HRF Inc., Raleigh, NC, USA), the samples were further diluted to target final concentrations of 0.9, 0.6, 0.3, 0.1, and 0 IU/mL and stored at −80°C. Testing of spiked sample material was performed on Technoclone Ceveron s100 using the Hyphen Biophen chromogenic FIX assay, calibrated with calibrators prepared with the N9-GP stock solution from step two and FIX-deficient plasma. The following N9-GP concentrations were measured: 0.90, 0.56, 0.29, and 0.10 IU/mL. Study sample sets were shipped on dry ice to the 11 study sites, with each laboratory receiving six sample sets—one for each day of the study and one as a reserve. Additionally, 30 sample sets remained stored at the Technoclone facility.

 

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Statistics

Data analysis was conducted using Excel 365 (Version 2408), R (Version 4.3.2), and MedCalc (version 20.011). In both the first and second surveys, assay variability (precision) was assessed by measuring all samples in triplicate over 5 consecutive days. Linear regression analysis was performed for each data series, and the conversion factor was determined based on the slope. This was first performed for samples from the first survey and separately for samples from the second survey. The coefficient of determination (R²) was used as an indicator of linearity and also showed the percentage of explained variability. The second survey served to investigate the long-term repeatability of results (‘proof of principle’) and, since it included one more N9-GP concentration than the first survey, it was used for the description of conversion factors in the results. Additionally, a Welch two-sample t-test and a two one-sided test (TOST) procedure were conducted to evaluate both the statistical differences and the equivalence of values obtained using the same instrument–reagent combinations across different laboratories.

 

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Results

Precision

The mean of the coefficients of variation (CV) of the two surveys are shown in [Table 3]. Notably, the overall CVs of all tested samples and those of the respective lowest samples (0.2 IU/mL in the first and 0.1 IU/mL in the second survey) were generally comparable.

Table 3 Precision

			CV survey #1 (%)		CV survey #2 (%)
Lab #	Reagent	Platform	All samples	0.2 IU/mL	All samples	0.1 IU/mL
1	Actin FS	CS-5100/2500[*]	7.7	5.5	2.8	2.7
2	Actin FS Biophen (chromo)	COAG360	4.9 5.9	5.7 7.9	4.9 7.8	6.1 4.7
3	Actin FS	CS-5100	7.7	5.5	8.1	9.3
4	Actin FS Biophen (chromo)	CS-5100	6.1 3.2	7.4 1.1	4.5 5.8	7.2 8.4
5	Actin FS Biophen (chromo)	CS-5100	13.6 9.3	14.6 7.7	8.5 5.0	10.7 5.0
6	SynthASil	ACL 500	3.9	5.1	3.9	3.4
7	STA C.K. Prest Biophen (chromo)	STAM	7.2 5.7	10.4 9.3	10.1 4.9	10.4 4.4
8	SynthaFAx	ACL 350	2.9	2.5	5.7	5.9
9	Pathromtin SL Biophen (chromo)	CS-5100	5.3 8.9	8.5 11.3	7.6 8.7	8.4 8.8
10	Pathromtin SL Biophen (chromo)	COAG360	14.2 n.p.	10.9 n.p.	4.3 7.6	5.8 7.5
11	Pathromtin SL Biophen (chromo)	BCSXP	n.p. 6.0	n.p. 6.8	7.1 9.2	5.5 14.1

Abbreviations: chromo, chromogenic; N9-GP, nonacog beta pegol; n.p., not performed.

Notes: The mean values of the coefficients of variation (CV) for all investigated N9-GP samples and the CV of the lowest N9-GP sample are presented. In both the first and second surveys, assay variability was assessed by measuring all samples in triplicate over 5 consecutive days.

^* Laboratory #1 used the CS-5100 in the first and the CS-2500 instrument in the second survey, respectively.

 

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N9-GP Recovery

When considering the mean recovery of all study test samples in both the first and second measurement series, N9-GP recovery differed substantially among all study test samples in both surveys when using aPTT-based one-stage assays ([Fig. 1A–C], [Supplementary Table S1]). Actin FS, SynthASil, and STA C.K. Prest underestimated, SynthAFax mildly overestimated, and Pathromtin SL severely overestimated the true N9-GP concentration.

Fig. 1 N9-GP recovery in the first and second surveys. The laboratory numbers are shown in x-axes (first vs. second survey, respectively). (A) Actin FS (blue bars; laboratories #1–5). (B) SynthASil (orange bars; laboratory #6), STA C.K. Prest (black bars; laboratory #7), SynthAFax (grey bars; laboratory #8). (C) Pathromtin SL (green bars; laboratories #9–11). (D) Hyphen Biophen chromogenic FIX (red bars, laboratories #2, 4, 5, 7, 9, 10, 11; laboratory #10 performed the chromogenic assay only in the second measurement series).

A high agreement of N9-GP recovery between the laboratories using Actin FS reagent (5/11 laboratories) was obtained ([Fig. 1A]). In contrast, the recovery of N9-GP differed significantly between the three laboratories that used Pathromtin SL, indicating a potential additional effect of the platform used (laboratory #9 CS-5100, #10 Atellica COAG360, #11 BCS XP, p < 0.001 for each comparison). The six laboratories using the Hyphen Biophen chromogenic FIX assay consistently found acceptable recoveries among all N9-GP concentrations with this assay ([Fig. 1D], [Supplementary Table S2]). Statistical analysis of values obtained from different laboratories using the same reagent–instrument combinations (Actin FS–CS-5100, Biophen–CS-5100, Biophen–COAG360) demonstrated statistically significant equivalence.

The recoveries of the lowest sample (0.2 IU/mL in the first and 0.1 IU/mL in the second survey, respectively) were found to be very similar to mean recoveries across all samples in all reagent–instrument combinations ([Supplementary Table S1]).

 

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Linear Regression Analysis Between N9-GP Concentrations and aPTT-Based Assays

Linear regression analysis revealed a strong and significant linear relationship between the N9-GP concentration and the mean values of all aPTT-based FIX activity measurements. This finding held true for all reagents in all laboratories for both the first (data not shown) and second surveys ([Table 4], [Fig. 2]).

Table 4 Linear regression analysis of aPTT-based FIX assays (y) and N9-GP activity (x) of the second survey for each laboratory

Lab #	Reagent	Platform	Intercept (β0)	Slope (β1)	R2	p
1	Actin FS	CS–2500	0.68	0.26	0.992	<0.001
2	Actin FS	COAG360	0.77	0.29	0.993	<0.001
3	Actin FS	CS-5100	0.83	0.32	0.994	<0.001
4	Actin FS	CS-5100	0.72	0.33	0.992	<0.001
5	Actin FS	CS-5100	0.91	0.34	0.992	<0.001
6	SynthASil	ACL 500	1.43	0.53	0.985	<0.001
7	STA C.K. Prest	STAM	−0.47	0.80	1.000	<0.001
8	SynthaFAx	ACL 350	6.94	1.26	0.979	0.001
9	Pathromtin SL	CS-5100	−8.32	6.29	0.998	<0.001
10	Pathromtin SL	COAG360	2.12	14.73	0.999	<0.001
11	Pathromtin SL	BCSXP	50.21	23.05	0.985	0.001

Fig. 2 Linear regression analyses of FIX activity (%) (y) and N9-GP concentration (%, where 100% = 1 IU/mL) (x) based on data from the second survey. Mean values of all N9-GP concentrations across both surveys are shown. Thin black dashed lines represent 95% confidence intervals of the regression functions. For laboratories #7 and #10, where unsatisfactory long-term repeatability was observed, the regression analysis of data from the first survey is also shown, denoted by an additional colored dashed line.

 

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Conversion Factors and Long-Term Repeatability

Based on the linear regression analysis, reagent-/platform-specific conversion factors were calculated as follows for both surveys: 1/slope (β1) ([Table 5]).

Table 5 aPTT-based FIX activity to N9-GP activity conversion factors in the first and second surveys

Lab #	Reagent	Platform	1st survey	2nd survey	Difference (%)
1	Actin FS	CS-5100/CS-2500*	3.34	3.85	14
2	Actin FS	COAG360	4.34	3.45	−19
3	Actin FS	CS-5100	3.34	3.13	−6
4	Actin FS	CS-5100	2.66	3.03	15
5	Actin FS	CS-5100	3.10	2.94	−4
6	SynthASil	ACL 500	1.92	1.89	−3
7	STA C.K. Prest	STAM	2.35	1.25	−47
8	SynthaFAx	ACL 350	0.91	0.79	−13
9	Pathromtin SL	CS-5100	0.19	0.16	−16
10	Pathromtin SL	COAG360	0.18	0.07	−62
11	Pathromtin SL	BCSXP	0.05	0.04	−5

Note: *Laboratory #1 used the CS-5100 in the first and the CS-2500 instrument in the second survey, respectively.

We applied a 20% variation cut-off for conversion factors as acceptability criteria for long-term repeatability within 3 years. All laboratories, except for laboratories #7 (STA C.K. Prest) and #10 (Pathromtin SL), met these criteria. During the second survey, laboratory #7 measured markedly higher values ([Figs. 1B] and [2]), which can partly be explained by the fact that two of the three replicates on one of the five measuring days were inexplicably high ([Supplementary Table S3]). In laboratory #10, results of the second measurement series were strongly and systematically higher than in the first one ([Figs. 1C] and [2], [Table 5]).

 

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Discussion

FIX activity monitoring in patients receiving pdFIX and rFIX agents is well established by either OSA or CSA. However, the monitoring of EHL formulations is challenging, as the modifications of the administered FIX (albumin/Fc-fusion, glycoPEGylation) have the potential to interact with components of aPTT reagents used in OSA. Previous studies evaluated the use of various aPTT reagents, demonstrating a broad range of data inconsistency.

When using the Actin FS reagent (ellagic acid as activator; Siemens, Marburg, Germany) to analyze N9-GP samples on CSB100i (optical clot detection; Sysmex, Kobe, Japan) or BCS XP (optical clot detection; Siemens) analyzers, Bowyer et al reported underestimation in low to high N9-GP concentrations (0.2, 0.6, 0.9 IU/mL) and overestimation in the very low concentration ranges (0.03 IU/mL).[10] Duboscq et al[31] reported overestimation of average recovery of four samples (0.05, 0.13, 0.5, 1.0 IU/mL) when measured with the same aPTT reagent on CS2500 (optical clot detection; Sysmex), ACL TOP500 (optical clot detection; IL, Bedford, MA, USA), and STA Compact (mechanical viscosity-based clot detection; Diagnostica Stago, Asniéres sur Seine, France) platforms. Divergent results were also found for the STA C.K. Prest reagent (kaolin as activator; Diagnostica Stago): Duboscq et al[31] reported overestimation when measured on a STA Compact instrument, while Persson et al[26] described underestimation (instrument information not provided). In addition, for SynthAFax (IL) and other aPTT reagents discrepant results were described (see [Table 1] for details). Besides, the External quality Control of diagnostic Assays and Tests (ECAT) foundation lists over 60 different available aPTT reagents from 29 different manufacturers[34] and for the vast majority, published data about the general suitability for EHL monitoring do not exist.

As previously described, we confirmed the underestimation of N9-GP recovery for Actin FS[10] [26] [35] and SynthAsil[10] [26] [31] assays, as well as the strong overestimation for the Pathromtin SL test.[10] [31] Within the results generated by using the STA C.K. Prest reagent, in which divergent results were described,[26] [31] we found underestimation in the first and a borderline acceptable recovery in the second survey. The recovery of N9-GP using the IL SynthAFax test was borderline acceptable in the first survey and mildly overestimated in the second survey.

Duboscq et al demonstrated that calibration of OSA with N9-GP-specific calibrators leads to an acceptable recovery of N9-GP with all of the 10 tested aPTT reagents (polyphenol, ellagic acid, and silica as activators).[31] In the present study, we showed that a linear relationship between the N9-GP concentration and the individual OSA test results exists for all aPTT reagents. Thus, an assay-/instrument-dependent conversion of OSA results to N9-GP activity using the corresponding regression equation seems feasible. In N9-GP monitoring, low trough values are of particular interest. Importantly, in this context, we demonstrated that recoveries of the lowest samples (0.2 IU/mL in the first and 0.1 IU/mL in the second survey, respectively) are comparable to those across the entire measurement range in all reagent–instrument combinations tested ([Supplementary Table S1]), indicating that back-calculation might be performed even in low N9-GP activities. Over a period of 3 years, the variation in conversion factors was low in 9 out of 11 laboratories. Thus, when performing back-calculation in real patient samples, a N9-GP-spiked sample with a known concentration should be measured simultaneously to ascertain the actual calibration and current conversion. A possible limitation of our study is that we established conversion factors using in-vitro N9-GP-spiked samples rather than ‘real’ post-infusion patient samples. However, Sørensen et al demonstrated that N9-GP-spiked plasma behaves similarly to post-administration clinical samples when using aPTT-based (SynthAFax) and chromogenic (Hyphen) FIX activity assays, as shown by comparable regression lines for in vitro and in vivo sample analyses.[24] Additionally, based on our data (which include high overestimation, partially unsatisfactory long-term repeatability when not calibrated daily, and the possible impact of the instrument used), using the Pathromtin SL reagent to back-calculate N9-GP activity seems particularly challenging. However, it can be used in laboratories with extensive experience in blood coagulation testing after determining an actual conversion factor, as recommended for all aPTT reagents used. It is noteworthy that commercial N9-GP calibrators are still not available; however, it may be possibly supplied in the future by Technoclone (Vienna, Austria). In addition, medical laboratories in the European Union are obliged to accurately validate this procedure, since it represents an in-house method according to the IVDR 2017/746 regulation.[36]

In conclusion, we could show that assay-/instrument-specific conversion factors might allow for back-calculation of aPTT-based FIX-activity results to N9-GP activities. Every laboratory should periodically determine its own conversion factor similar to the approach in this study. Suitable N9-GP control material must be used each time when a real patient sample is measured to ascertain the validity of the actual calculation. This method might also serve as ‘back-up’ in cases where chromogenic assays are not available for any reasons (e.g., due to rather short reagent durability of those rarely used assays).

We believe that our results provide practical information for medical laboratories to overcome the issue of correctly measuring FIX activity with FIX OSA assays in HB patients treated with N9-GP.

 

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Conflicts of Interest

Haushofer A: Research grant support from ÖGLMKC (Austrian Society for Laboratory Medicine and Clinical Chemistry) for conducting this study. Consultancy and speaker fees from Novo Nordisk, Baxter, Shire, Takeda, Sysmex, Siemens Diagnostics, Roche Diagnostics, Sobi, and Sanofi-aventis.

Prüller F: Speaker fees from Sysmex and Novo Nordisk.

Lirk G: Fee note for the statistical evaluation of data (Novo Nordisk).

Acknowledgements

The authors thank Novo Nordisk A/S, Bagsværd, Denmark, Precision BioLogic, Inc, and Nikolaus Binder (Technoclone GmbH, Vienna, Austria) for supplying the N9-GP spiked samples.

Author Contributions

The potential linear relationship between the measured values, the interim reports prepared for the study participants, and the basis of the evaluation were developed and documented by AH. Under supervision of AH all authors contributed to the conception and design of the study. All authors except GL coordinated sample measurements in their respective laboratories. GL performed statistical analyses. CI wrote the original draft, and all other authors commented on the manuscript. All authors agree to be accountable for all aspects of the work. All authors read and approved the final manuscript.

Ethical Approval

Not applicable.

Consent to Participate

Not applicable.

Availability of Data and Materials

Data available on request from the authors.

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Publication History

Received: 22 January 2025

Accepted: 05 May 2025

Article published online:
29 August 2025