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Keywords:

  • assay discrepancies;
  • recombinant B-domain deleted FVIII;
  • standardization

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Participating laboratories
  6. Provided materials
  7. Study procedures
  8. Data analysis
  9. Results
  10. Assay reagents used by participating laboratories
  11. Results
  12. Effect of assay reagents
  13. Discussion
  14. Acknowledgements
  15. References

Summary.  Advances in production technologies of factor (F)VIII concentrates during the last two decades has resulted in very pure and safe products. In assessment of recombinant FVIII:C, inconsistent assay values are found comparing one-stage assays with two-stage (e.g. amidolytic) methods. Such discrepancies have been quite prominent in the case of a B-domain deleted recombinant FVIII (BDDrFVIII, ReFacto®). In order to alleviate this assay variance, a product-specific reference standard [the ReFacto Laboratory Standard™ (RLS)], was established for laboratory use with either one-stage clotting or chromogenic substrate assays for the measurement of FVIII:C in ReFacto-containing patient samples. The primary objective of the current study was to assess, under field laboratory conditions, the accuracy and precision of the one-stage clotting assay for the determination of FVIII:C in ReFacto-containing samples employing the new concentrate standard. A secondary goal was to assess whether use of the RLS would minimize the discrepancy between one-stage clotting and chromogenic substrate assays. Thirty-one clinical laboratories worldwide participated in the study of severe-hemophilic plasma (SHP) samples that had been spiked with ReFacto to target levels of 0.9, 0.6 and 0.2 IU mL−1. FVIII:C levels were determined against both the RLS and the local in-house plasma standard (IHS). The results showed good agreement between laboratories in FVIII:C levels obtained by one-stage clotting assays utilizing the RLS, and a good degree of accuracy was found compared with the intended target values. Consistent with previously published data, a discrepancy of approximately 30% was observed between one-stage clotting and chromogenic potencies when the IHS was used as the calibrator. The discrepancy between one-stage and chromogenic assay methodologies was significantly reduced when the RLS was employed as calibrator in the one-stage assay. In conclusion, the study demonstrates that accurate and precise FVIII:C results can be obtained for ReFacto-containing SHP samples by clinical laboratories using a product-specific standard in one-stage clotting assays. In addition, the product-specific reference standard significantly reduced the discrepancy between the one-stage clotting and the chromogenic substrate assay for ReFacto.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Participating laboratories
  6. Provided materials
  7. Study procedures
  8. Data analysis
  9. Results
  10. Assay reagents used by participating laboratories
  11. Results
  12. Effect of assay reagents
  13. Discussion
  14. Acknowledgements
  15. References

Currently, both one-stage clotting and chromogenic substrate assays are utilized in the diagnosis and treatment of patients with hemophilia A, as well as in the assessment of factor (F)VIII concentrate potency. Despite concerted global efforts towards standardization, considerable discrepancies are still observed between one-stage and chromogenic results [1–3]. When high-purity FVIII concentrates are assessed, the potency measured by one-stage assays may be up to 30% higher than that measured by chromogenic assays when assayed against a concentrate reference standard [3]. For full-length recombinant FVIII (rFVIII) products, the one-stage potency has been shown to be approximately 80% of the potency determined using a chromogenic substrate assay [4]. For B-domain depleted recombinant FVIII (BDDrFVIII, ReFacto) the potency, as determined by one-stage clotting assays using common commercial activated partial thromboplastin time (APTT) reagents, is consistently low, with values decreased by 20–50% of the chromogenic substrate activity having been reported [4,5].

A further complication regarding the measurement of FVIII:C is the inherent variability associated with the testing methods. The extent of variability between methods and between laboratories creates problems with respect to the standardization and harmonization of the assessment of FVIII:C. A number of systematic studies have been performed attempting to evaluate the measurement of FVIII:C, and resulted in the development of the following recommendations designed to standardize the assessment of FVIII:C in concentrates [3]:

  • 1
    A concentrate standard calibrated against the current World Health Organization (WHO) concentrate standard should be used.
  • 2
    Both test and standard concentrate should be prediluted in severe hemophilia A plasma.
  • 3
    All assay buffers should contain 1% albumin.
  • 4
    The chromogenic substrate method should be employed as the reference method.

Subsequent studies have demonstrated a direct correlation between adherence to the concentrate testing recommendations and good interlaboratory agreement of FVIII potency measurements [6]. For the full-length rFVIII products, an additional factor that has played a role in reducing discrepant potency values between one-stage and chromogenic testing has been the establishment of the WHO 6th International Standard for Blood Coagulation FVIII:C (WHO 6th IS, 97/616), prepared from recombinant full-length FVIII. By nature of a similar molecular composition, assessment of rFVIII potency vs. the WHO 6th IS, by default, follows the biometric principle of ‘like vs. like’ and should allow for accurate potency determinations.

Nevertheless, the monitoring of FVIII therapy in clinical laboratories continues to suffer from the above-described assay discrepancy and FVIII assay variability. Accurate determination of circulating FVIII:C levels is paramount to patient care, as underestimation may lead to increased dosing and elevated treatment costs.

Postinfusion plasma samples from patients have demonstrated inconsistent FVIII:C levels following administration of either high-purity plasma-derived FVIII [7] or recombinant factor products to hemophilia A patients [8].

Despite these issues, global recommendations for the assessment of FVIII:C activity in postinfusion samples have yet to be established. Interestingly, potential recommendations under discussion [6] involve adherence to the ‘like vs. like’ principle for measuring biologics and have been demonstrated to be successful for the evaluation of rFVIII in an in vivo recovery study [9]. With the existing two full-length rFVIII products labeled on the basis of the one-stage assay (the most prevalent method used clinically for assessing FVIII:C in postinfusion samples) and the WHO 6th IS comprised of rFVIII, discrepant potency values between the one-stage and chromogenic assay for rFVIII are expected no longer to be prevalent amongst testing laboratories.

The previous accommodations have not proven to resolve method-based potency discrepancies for B-domain deleted FVIII (ReFacto). The situation with ReFacto is complicated by two factors: (i) in general, the discrepancy between one-stage and chromogenic results for ReFacto is greater than that observed for full-length rFVIII, and (ii) in accordance with the European Pharmacopoeia reference method for assessing the potency of FVIII concentrates, the potency of ReFacto is assigned using a chromogenic substrate assay. With the prevalent method used to monitor FVIII replacement therapy in the clinical setting remaining the one-stage clotting assay, assessing samples from patients receiving ReFacto for FVIII:C may lead to difficulties in the clinical setting. Potency determinations of vial content may also prove problematic due to the compositional difference between the WHO 6th IS (97/616, comprised of full-length rFVIII) and the ReFacto product (BDDrFVIII). The relationship between rFVIII and BDDrFVIII with respect to thrombin activation profiles [10] and thrombin generation [11] has been explored elsewhere and may provide some insight as to why discrepant potency values may be obtained for ReFacto when assessed vs. a non-product-specific standard (WHO 6th IS).

Previous investigations have shown that method-based potency discrepancies for postinfusion estimation of FVIII may be minimized if (i) a chromogenic assay is employed for FVIII quantification, or (ii) the working standard used in the one-stage FVIII assay consists of the same concentrate infused (i.e. ‘like vs. like’), rather than a normal plasma standard [9,12]. Therefore, the use of a product-specific reference standard for ReFacto is consistent with the biometric principle of ‘like vs. like’ recommended by the Factor VIII and Factor IX Scientific and Standardization Subcommittee (SSC) of the International Society on Thrombosis and Haemostatsis (ISTH) to reduce discrepancies among assays, products, standards and laboratories, and should provide a means of minimizing method-based potency discrepancies for ReFacto. The primary aim of the current study was to assess the accuracy and precision of the one-stage assay under true field conditions of using a ReFacto concentrate standard [ReFacto Laboratory Standard (RLS)] for determining the FVIII:C level in severe-hemophilic plasma (SHP) samples containing ReFacto in laboratories providing hemophilia service worldwide.

Participating laboratories

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Participating laboratories
  6. Provided materials
  7. Study procedures
  8. Data analysis
  9. Results
  10. Assay reagents used by participating laboratories
  11. Results
  12. Effect of assay reagents
  13. Discussion
  14. Acknowledgements
  15. References

Thirty-three laboratories worldwide were invited to participate in the field study and received field study kits based on their declared interest. All laboratories performed FVIII:C assays on provided materials using their routinely used standard reagents, and instrumentation.

Provided materials

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Participating laboratories
  6. Provided materials
  7. Study procedures
  8. Data analysis
  9. Results
  10. Assay reagents used by participating laboratories
  11. Results
  12. Effect of assay reagents
  13. Discussion
  14. Acknowledgements
  15. References

Field study test kits were shipped to participants by George King Bio-Medical (Overland Park, KS, USA), containing the ReFacto Laboratory Standard (9.4 IU per vial) and three ReFacto-spiked plasma samples (S1, S2 and S3). The study samples were prepared from commercial ReFacto drug substance and diluted to the target concentrations using congenital SHP (George King Bio-Medical). Each laboratory supplied their own routine in-house FVIII standard (IHS), FVIII-deficient plasma and APTT reagents for use in the one-stage clotting assay, as well as chromogenic kits if routinely used. Laboratories were instructed to predilute the ReFacto Laboratory Standard in FVIII-deficient plasma containing normal functional levels of von Willebrand factor (VWF):Ag (at ≥ 0.5 IU mL−1).

Paper and electronic assay reporting forms were provided for the collection of assay data, final calculated FVIII:C concentrations, along with information on assay methodology and conditions. Data were collected between November 2001 and March 2002.

Study procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Participating laboratories
  6. Provided materials
  7. Study procedures
  8. Data analysis
  9. Results
  10. Assay reagents used by participating laboratories
  11. Results
  12. Effect of assay reagents
  13. Discussion
  14. Acknowledgements
  15. References

Laboratories were instructed to assay samples on four separate days and calculate the FVIII:C levels of each sample against both RLS and IHS calibration curves. Test kits contained sufficient supplies such that two sets of fresh samples could be used for each run, with samples thawed or reconstituted immediately prior to assay. It was expected that fresh standard curves would be prepared with each assay and that at least three dilutions would be performed on each sample. Instructions were provided for predilution of the RLS from 9.4 IU mL−1 to 1.0 IU mL−1 using a FVIII-deficient plasma containing normal functional levels of VWF. Laboratories were instructed to follow their routine laboratory practices if they could not conform to an aspect of the field study protocol, and deviations from the protocol were specified in the information returned for data analysis. Laboratories evaluated samples using either a one-stage clotting assay, a chromogenic assay, or both if routinely performed. A balanced assay design was employed to prevent sample placement bias. Laboratories provided raw data (clotting times for one-stage assays; optical density values for chromogenic assays) and calculated potencies. The calculated potencies from individual laboratories were used for all calculations. Each laboratory provided information on whether they computed FVIII:C potencies by graphical or computer analysis and whether they used parallel line or linear regression.

Data analysis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Participating laboratories
  6. Provided materials
  7. Study procedures
  8. Data analysis
  9. Results
  10. Assay reagents used by participating laboratories
  11. Results
  12. Effect of assay reagents
  13. Discussion
  14. Acknowledgements
  15. References

All results were submitted to a central, independent processing site that entered, reviewed and analyzed all data. All manual data entry was checked against source documentation for 100% accuracy. Files submitted electronically were saved unedited with a working copy made for analysis and 10% of all electronic data was checked against printouts of the original electronic data file to ensure accuracy. Laboratories were queried to clarify improperly completed forms or data that appeared erroneous to minimize the possibility of data discrepancies. Descriptive statistics were performed separately on FVIII:C levels obtained by one-stage clotting and chromogenic assays. Statistical analyses included minimum and maximum values, inter- and intralaboratory means, standard deviation and relative standard deviation. Intralaboratory statistics were calculated from the FVIII:C levels obtained on each of the four assay days. Results were stratified by laboratory and sample: FVIII:C concentration, percent of mean values, percent of target values, and inter- and intra-assay precision were calculated.

Assay reagents used by participating laboratories

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Participating laboratories
  6. Provided materials
  7. Study procedures
  8. Data analysis
  9. Results
  10. Assay reagents used by participating laboratories
  11. Results
  12. Effect of assay reagents
  13. Discussion
  14. Acknowledgements
  15. References

Thirty-one laboratories (94%) returned study data; 30 laboratories submitted one-stage assay results; six laboratories provided both chromogenic and one-stage assay results; one laboratory provided only chromogenic assay results. As shown in Table 6, 14 different APTT reagents were used by participating laboratories. Fourteen different FVIII-deficient plasmas and 12 different instrument systems were used in the study. Thus there was no common reagent–instrument combination. In-house calibrators were used by eight laboratories, with the number of donors collected in the pool ranging from > 20 to 200. Calibration of in-house plasma was performed in various ways, including: (i) comparison with a WHO or National Institute for Biological Standards and Control (Potters Bar, UK) standard, (ii) comparison with the previous in-house standard; or (iii) assigned value of 100%. Remaining laboratories used a commercial plasma calibrator with a known assigned value. Commercial calibrators were from at least nine different manufacturers. The specific manufacturer was not reported by seven laboratories. Three different assay kit types were used by the seven laboratories reporting chromogenic results. Of the laboratories performing one-stage clotting assays, only 13% (4/30) included 1% albumin in the assay buffer vs. 86% (6/7) of the laboratories performing chromogenic substrate assays.

Table 6.   Mean factor (F)VIII levels by activated partial thromboplastin time (APTT) reagent
APTT reagentNo. of labsCalculated vs. RLS (IU mL−1)Calculated vs. IHS (IU mL−1)
S1S2S3S1S2S3
  1. RLS, ReFacto Laboratory Standard; IHS, in-house plasma standard.

Actin10.790.560.250.550.400.18
Actin FS30.830.560.210.700.480.18
Actin FSL30.940.690.290.800.570.24
IL—undefined20.840.600.240.760.530.21
IL APTT silica30.720.530.210.660.470.18
IL APTT-SP20.830.580.220.690.490.19
In house: 0.75% aluminum silicate;10.880.620.250.680.470.18
 Bell & Alton phospholipid
O.T. Platelin LS50.860.590.220.560.400.15
O.T. Auto APTT30.780.550.210.680.450.16
Stago CK Prest with cephalin 1/2010.800.580.230.790.550.22
Stago PTT-A10.960.710.250.710.480.18
Stago—undefined10.860.610.240.580.410.16
Synthasil30.850.570.230.650.440.17
Thrombosil10.860.610.230.600.440.17

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Participating laboratories
  6. Provided materials
  7. Study procedures
  8. Data analysis
  9. Results
  10. Assay reagents used by participating laboratories
  11. Results
  12. Effect of assay reagents
  13. Discussion
  14. Acknowledgements
  15. References

The three study samples, S1, S2 and S3, contained ReFacto at concentrations of 0.9, 0.6 and 0.2 IU mL−1, respectively. Individual laboratory means of four replicate determinations using the one-stage clotting assay for S1 (0.9 IU mL−1) are shown using the RLS (Fig. 1A) and against the IHS (Fig. 1B).

image

Figure 1.  Distribution of S1 (0.9 IU mL−1) factor (F)VIII:C results by laboratory using ReFacto Laboratory Standard (RLS) (A) and in-house plasma standard (IHS) (B). Each square represents a laboratory whose S1 FVIII:C level was within the 0.05 IU mL−1 range depicted on the abscissa.

Download figure to PowerPoint

The graphs of Fig. 1 show an increase in the FVIII:C levels when the RLS was used as the reference standard compared with results obtained vs. a plasma-based standard. A similar, although less pronounced shift was seen with S2 and S3, as shown in Table 1. This table represents the overall means and standard deviations for samples assessed using the one-stage assay.

Table 1.   Mean one-stage factor (F)VIII:C levels
 Calculated vs. RLS (IU mL−1) n = 30Calculated vs. IHS (IU mL−1) n = 30
S1S2S3S1S2S3
  1. Mean FVIII:C levels were calculated from the mean of the results of the four individual assay days from all laboratories. n, Number of laboratories that submitted results for each sample; RLS, ReFacto Laboratory Standard; IHS, in-house plasma standard.

Mean 0.85 0.59 0.23 0.67 0.47 0.18
SD 0.11 0.08 0.04 0.10 0.07 0.04
% CV12.8%13.3%17.0%15.0%15.6%20.1%
Min. 0.57 0.47 0.17 0.52 0.36 0.12
Max. 1.17 0.88 0.39 0.93 0.69 0.30
Target 0.9 0.6 0.2 0.9 0.6 0.2

The overall means and standard deviations for the chromogenic substrate assays are presented in Table 2.

Table 2.   Mean chromogenic factor (F)VIII levels
 Calculated vs. RLS (IU mL−1) n = 4Calculated vs. IHS (IU mL−1) n = 7
S1S2S3S1S2S3
  1. Mean FVIII:C levels were calculated from the mean of the results of the four individual assay days from all laboratories. n, Number of laboratories that submitted results for each sample; RLS, ReFacto Laboratory Standard; IHS, in-house plasma standard.

Mean 0.97 0.65 0.23 0.98 0.66 0.25
SD 0.11 0.07 0.03 0.12 0.08 0.03
% CV11.1%11.3%11.4%12.0%12.6%11.4%
Min. 0.82 0.54 0.20 0.80 0.54 0.20
Max. 1.06 0.71 0.26 1.11 0.73 0.27
Target 0.9 0.6 0.2 0.9 0.6 0.2

As seen in other studies using concentrate-specific standards [4], the differences between the chromogenic and one-stage results were reduced considerably with the use of RLS. When one-stage clotting results were compared as a percent of the chromogenic substrate assay values, where both methods were calibrated using the RLS, FVIII:C levels were 88%, 92% and 100% for S1, S2 and S3, respectively, of target values. In contrast, when both assays were calibrated using the in-house plasma standard, comparison of the one-stage clotting as a percent of the chromogenic method demonstrated FVIII:C values that were 68%, 71% and 73% for S1, S2 and S3, respectively.

The percent of target values for the one-stage and chromogenic assays using RLS and IHS are shown in Tables 3 and 4, respectively. The FVIII:C levels using RLS as the standard in the one-stage assay approximated target values (94%, 99% and 116%) more closely than values obtained using IHS calibration (74%, 78% and 90%), which were 10–26% lower overall. There were no significant differences between RLS and IHS results using the chromogenic assay for S1, S2 or S3.

Table 3.   Percent of target values—one-stage assay
 Calculated vs. RLS (%) n = 30Calculated vs. IHS (%) n = 30
S1S2S3S1S2S3
  1. RLS, ReFacto Laboratory Standard; IHS, in-house plasma standard; RSD, Relative Standard Deviation.

Mean, % 94 99116 74 78 90
SD, % 12.1 13.2 19.7 11 12 18
% RSD 12.8 13.3 17.0 15 16 20
Min., % 63 78 85 58 60 61
Max., %130147194104116153
Target  0.9  0.6  0.2  0.9  0.6  0.2
Table 4.   Percent of target values—chromogenic assay
 Calculated vs. RLS (IU mL−1) n = 4Calculated vs. IHS (IU mL−1) n = 7
S1S2S3S1S2S3
  1. RLS, ReFacto Laboratory Standard; IHS, in-house plasma standard; RSD, Relative Standard Deviation.

Mean, %108108115109109123
SD, % 11.9 12.3 13.1 13 14 14
% RSD 11.1 11.3 11.4 12 13 11
Min., % 92 91 99 89 89 99
Max., %117118132123122136
Target  0.9  0.6  0.2  0.9  0.6  0.2

There was a high degree of precision within a laboratory, with the average intralaboratory coefficient of variation (CV) at < 5%. The interlaboratory variability was generally acceptable (although there were a few laboratories with discordant results, Fig. 1A,B; Table 1), with CVs for the ReFacto test samples lower when assessed vs. the RLS compared with values obtained vs. the IHS (Table 5). Overall, the variability noted in this study compared favorably with other published field studies [2,12]. Factors that may play a role in interlaboratory variability for FVIII:C measurements include the choice of APTT reagent and VWF:Ag content of the FVIII-deficient plasma used for predilution of the RLS in the one-stage assay.

Table 5.   Inter-laboratory precision
MethodCalculated vs. RLS (% CV)Calculated vs. IHS (% CV)
S1S2S3S1S2S3
  1. RLS, ReFacto Laboratory Standard; IHS, in-house plasma standard.

Chromogenic, %11.111.311.412.012.611.4
One-stage, %12.813.317.015.015.620.1

Effect of assay reagents

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Participating laboratories
  6. Provided materials
  7. Study procedures
  8. Data analysis
  9. Results
  10. Assay reagents used by participating laboratories
  11. Results
  12. Effect of assay reagents
  13. Discussion
  14. Acknowledgements
  15. References

A variety of different APTT reagents and FVIII-deficient plasma were used in the one-stage clotting assay. The APTT reagents used varied in phospholipid composition and concentration, as well as contact activators for initiation of the coagulation cascade. These reagents, when utilized in an assay system calibrated with an in-house plasma standard, recovered levels that were 12–40% lower than target values (Table 6), which is consistent with previously reported data [4]. Although the dataset per APTT reagent is not large enough to perform a statistical evaluation, there is evidence for a bimodal distribution for results obtained vs. the IHS, with a cluster of results for sample S1 at 20% lower than the average for the other APTT reagents (0.56 vs. 0.70 IU mL−1, respectively). As most data for this low S1 dataset were obtained vs. a single reagent (Platelin LS; BioMérieux, Durham, NC, USA), the low values could reflect bias associated with this particular reagent. However, when used in conjunction with the RLS, the levels obtained were consistent with target values for all APTT reagents, illustrating the utility of using the RLS in the one-stage clotting assay for ReFacto-containing samples, regardless of APTT reagent choice.

The choice of FVIII-deficient plasma for predilution of the RLS may also be an important parameter in the one-stage assay, as those plasma deficient in VWF:Ag (< 0.1 IU mL−1) may result in prolonged clotting times for the RLS, with subsequent elevated values for the study samples. However, the majority of laboratories (23/30) in this study used either congenital FVIII-deficient plasma, immunodepleted or chemically depleted plasma known to contain normal functional levels of VWF:Ag [13] for their one-stage clotting assay. Interestingly, of the seven remaining laboratories, five used plasma known to be deficient in VWF and yet the potency values obtained for the study samples were not significantly different when compared with laboratories using plasma containing normal levels of VWF (two laboratories did not provide information regarding their choice of FVIII-deficient plasma). Despite these variables, the data clearly demonstrate that use of the RLS in the one-stage clotting assay allows for accurate recovery of ReFacto.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Participating laboratories
  6. Provided materials
  7. Study procedures
  8. Data analysis
  9. Results
  10. Assay reagents used by participating laboratories
  11. Results
  12. Effect of assay reagents
  13. Discussion
  14. Acknowledgements
  15. References

This field study was performed to assess both the accuracy and precision of the one-stage clotting assay for the determination of FVIII:C in ReFacto-containing samples using a ReFacto concentrate standard (ReFacto Laboratory Standard, RLS). A secondary goal was to evaluate whether use of the RLS would minimize method-based potency discrepancies for ReFacto in laboratories performing both one-stage clotting and chromogenic substrate assays.

The study results demonstrate one-stage assay values for the three ReFacto-containing plasma samples showing a high degree of accuracy when compared with the chromogenic target values; 94%, 99% and 116% for samples S1, S2 and S3, respectively, when assessed vs. the product-specific standard (RLS). Additionally, any observed discrepancy between the one-stage clotting and chromogenic methods was minimized (< 10%) when the RLS was used to standardize the one-stage assay vs. an in-house plasma standard used for the chromogenic assay. Methodological variables encountered in this field study, such as different APTT reagents and FVIII-deficient plasma, were minimized by calibration of the one-stage clotting assay with the RLS, illustrating the utility of the RLS for any laboratory monitoring of ReFacto patient samples, regardless of assay methodology used.

The data for this SSC field study correlate well with other SSC studies previously reported [2] and illustrate that use of the one-stage clotting assay, when calibrated with the RLS, provides an accurate and precise assessment of FVIII:C in ReFacto-containing plasma samples in clinical laboratories worldwide.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Participating laboratories
  6. Provided materials
  7. Study procedures
  8. Data analysis
  9. Results
  10. Assay reagents used by participating laboratories
  11. Results
  12. Effect of assay reagents
  13. Discussion
  14. Acknowledgements
  15. References

The ReFacto Field Study participants include: Rosella Bader, Centro Emofilia e Trombosi, Milano, Italy; Sandy Bohn, Karen Meyer, Fairview University Medical Center, Minneapolis, MN, USA; E. G. Bovill, Elizabeth Golden, Fletcher Allen Health Care, Burlington, VT, USA; Ulrich Budde, Labor Prof Arndt and Partner, Hamburg, Germany; Clive Burgess, Great Ormond Street Hospital for Children, London, UK; Claudine Caron, Hôpital Cardiologique, Lille, France; Paula Dunnington, Sheffield Children's Hospital, Sheffield, UK; Michele Flanders, A.R.U.P., Salt Lake City, UT, USA; Lorna Germain, Royal Infirmary of Edinburgh, Edinburgh, UK; Keith Hoots, D. Sayre, University Texas, Houston, TX, USA; J. Ingerslev, H. Larsen, University Hospital Skejby, Aarhus, Denmark; Cindy Johns, Esoterix, Inc., Aurora, CO, USA; H. Joist, D. Chance, St Louis University Hospital, St Louis, MO, USA; Lynn Hoernig, CORE Laboratory, Children's Hospital of Buffalo, Buffalo, NY, USA; Judith Meyer, Children's Hospital Medical Center, Oakland, CA, USA; J. Monteagudo, Hospital Clinico, Barcelona, Spain; Jane Needham, North Hampshire Hospital, Basingstoke, UK; Claude Negrier, Hôpital Edouard Herriott, Lyon, France; Joanna Nightingale, Kent and Canterbury Hospital, Canterbury, UK; Ulla Oswaldsson, Biovitrum AB, Stockholm, Sweden; E. Peerschke, Donna Castellone, New York Presbyterian Hospital, New York, NY, USA; Angus McCraw, Anne Riddell, Royal Free Hospital, London, UK; S. Kitchen, B. Sampson, Royal Hallamshire Hospital, Sheffield, UK; Inge Scharrer, University Hospital, Frankfurt, Germany; Midora Shima, Nara Medical University, Kashihara, Nara, Japan; Joan Tusell, Hospital Materno Infantil, Barcelona, Spain; Janis Warren, Leicester Royal Infirmary, Leicester, UK; Lisa Weber, Comprehensive Bleeding Disorder Center, Peoria, IL, USA; Jean Lusher, Carol R. Wiseman, Children's Hospital of Michigan, Detroit, IL, USA; Angela Quinlan, Children's Hospital of Eastern Ontario, Ottawa, Ontario, Canada; Linda Stang, Special Coagulation Laboratory, University of Alberta Hospital, Edmonton, Alberta, Canada, who are all thanked for their participation and cooperation. The authors thank Judy Gillissen for coordinating the assembly and distribution of the field study test kits.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Participating laboratories
  6. Provided materials
  7. Study procedures
  8. Data analysis
  9. Results
  10. Assay reagents used by participating laboratories
  11. Results
  12. Effect of assay reagents
  13. Discussion
  14. Acknowledgements
  15. References