SEARCH

SEARCH BY CITATION

Keywords:

  • automated VWF:RCo assay;
  • von Willebrand disease;
  • von Willebrand factor;
  • von Willebrand factor activity assay

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Summary. Background, objectives and methods: An accurate, sensitive and precise assay for reliable determination of the ristocetin cofactor activity of von Willebrand factor (VWF:RCo) in plasma and von Willebrand Factor (VWF)-containing concentrates has been evaluated. The assay is based on a commercially available automated protocol with modifications including a combination of adding additional ristocetin and the use of two calibration curves for the high and low measuring ranges. Results: Addition of extra ristocetin resulted in improved measurement of VWF recoveries from various VWF-containing concentrates that were underestimated using the standard automated protocol. The modifications resulted in improved assay performance over an extended measuring range (2.00–0.03 IU mL−1). Accuracy was tested using VWF deficiency plasma spiked with the 1st international standard (IS) for VWF concentrate. Seven dilutions, ranging from 1.80 to 0.05 IU mL−1, were analyzed and resulted in measured concentrations between 80% and 100% of the assigned potency of the standard. Linearity was determined from the regression plot of the same concentrate dilutions and resulted in a correlation coefficient of 0.998. The repeatability, expressed as coefficient of variation, was 2% in the normal range (0.90 IU mL−1) and 8% at the level of 0.05 IU mL−1. The corresponding reproducibility results were 2% and 15% at the normal and low measuring ranges, respectively. Conclusions: Analysis of patients with von Willebrand disease (VWD) indicates that the modified automated BCS® protocol has a superior discrimination power compared with the standard protocol. This is especially true in samples with low VWF, as in patients with type 3 VWD.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

von Willebrand disease (VWD) is the most common hereditary bleeding disorder with an overall prevalence of 0.1–1% and is caused by either a quantitative or qualitative deficiency of von Willebrand factor protein (VWF) [1,2]. The precise diagnosis of VWD is a challenge to clinicians and there are different types depending on the kind of underlying VWF deficiency. Type 1 is characterized by a partial quantitative defect; type 2 by qualitative defects independent of the amount of VWF antigen (VWF:Ag); and type 3 by a virtual absence of VWF. Type 2 can be subdivided further based upon the exact nature of the qualitative impairment [3]. Clinically, the manifestations of VWD may vary from striking to very subtle, thus many patients with mild symptoms may remain undiagnosed [4].

Laboratory assays should be able to detect the qualitative and/or quantitative deficiencies in VWF and to assess its functional activity and, are utilized clinically to evaluate individuals suspected of being affected with VWD, especially those with a personal or family history of excessive mucocutaneous bleeding. Over the few last decades, a number of laboratory tests have been developed, including the bleeding time; quantitative VWF:Ag measurements; qualitative analysis of the multimeric structure; and functional activity assessments of VWF, based on the use of the glycoprotein antibiotic ristocetin sulphate (VWF:RCo), which agglutinates normal platelets in the presence of VWF. The clinical value of these assays is in part limited by the uncertainty of their specificity and sensitivity, as corroborated in the recently published American guidelines [5]. These same assays are also utilized in the quality control and potency determinations of manufactured replacement products and in the prediction of their hemostatic effectiveness when administered to prevent or reverse the hemorrhagic complications of VWD.

The major drawback of the VWF assays is the variability of results, often predicated on the technical experience of laboratory personnel, the assay techniques/commercial reagents, kits utilized and the equipment platforms employed [3,6]. VWF:RCo measures the interaction of VWF with the platelet receptor glycoprotein Ibα, which ultimately induces platelet agglutination [7]. This assay permits the distinction between different VWD subtypes and is widely used to monitor treatment when a diagnosis has been confirmed [8].

The manual VWF:RCo agglutination assay has been recommended as the assay of choice for assessing VWF functional activity according to the European Pharmacopoeia. VWF:RCo can also be measured via a semi-automated method in which platelet agglutination is quantified using optical aggregometry with subsequent subjective evaluation of the slope of the reaction compared with a standard curve (steepest slope). This assay has been shown to have a high variability depending on the reagents and standards used, which makes it less reliable as well as labor and cost intensive [9–11]. Thus, in recent years, several fully automated VWF:RCo assay protocols have been described [11–15]. They allow greater throughput of samples with improved analytical precision compared with manual and semi-automated assays.

Despite the clear advantages of modern automated VWF:RCo methods, there are still notable limitations, such as high limits of detection (LODs), underestimation of activities and variable results depending on the type of analyzer and reagent lots used. For the pharmacokinetic analysis of samples, especially from type 3 VWD patients who have very low VWF:RCo levels, a high sensitivity in the low concentration range is required. Furthermore, the automated assay in its original form does not seem suited for estimation of samples of VWF-containing coagulation factor concentrates. Compared with current international standards, potencies of these concentrates were grossly underestimated with the current standard automated assay, irrespective of the type or brand of concentrate. This was not the case when these same comparative studies were conducted using the manual assay.

In the present study, we present an adaptation of the automated VWF:RCo protocol, which shows additional improved sensitivity and precision, especially in plasma samples from patients with low levels of VWF as found with severe type 3 VWD. Additionally, the modified assay is better suited for plasma samples containing VWF/FVIII concentrates.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Samples and standards

The 1st International Standard (IS) for VWF concentrate (00/514) and the 5th IS for FVIII and VWF in plasma (02/150) were obtained from the National Institute for Biological Standards and Control (NIBSC, Potters Bar, UK). Immunodepleted VWF-free plasma (VWFDP) was produced by Hyphen BioMed (Neuville-sur-Oise, France). Ristocetin was purchased from Mascia Brunelli S.p.A., Milan, Italy. Determination of the ristocetin concentration in the BC® VW reagent was performed by Chemcon GmbH (Technisches Büro für technische Chemie, Vienna, Austria) using high-performance liquid chromatography (HPLC) analysis. Plasma samples from seven VWD type 2 (six 2A and one 2B) and seven type 3 patients as diagnosed by the treating physician were collected after a minimum of 7 days washout of any treatment. The laboratory diagnosis of VWD type 2 included a combination of VWF:Ag and VWF:RCo where the latter was disproportionally reduced and a lack of high-molecular-weight multimers. Type 3 VWD was confirmed by the absence of VWF:Ag, VWF:RCo and VWF multimers. In none of these cases the automated protocols for VWF:RCo (see below) were used for diagnosis. The plasma was frozen and stored at −70 °C prior to testing, and left at room temperature for ≤ 1 h after thawing just before running the below assays. The patient samples were obtained with the approval of local Institutional Review Boards and all plasma samples were anonymized to protect patient confidentiality.

Automated VWF:RCo assays

The standard automated protocol using the BC® VW reagent (Siemens Healthcare Diagnostics, Marburg, Germany) was carried out on the coagulation analyzer BCS XP® [Siemens Healthcare Diagnostics (formerly DadeBehring)] and agglutination was measured at 570 nm, according to the manufacturer’s instructions. One vial of the BC® VW reagent, containing lyophilized platelets, ristocetin and ethylenediaminetetraacetic acid (EDTA), was reconstituted with 4 mL of distilled H2O before use. In short, the assay involved mixing 20 μL of 0.9% NaCl, 10 μL of plasma sample and 150 μL of BC® VW reagent, and agglutination was then measured (see Table 1 for details). Plasma samples were analyzed in duplicate, and dilutions of the calibration samples were analyzed in quadruplicate as recommended by the manufacturer. Physiological saline (0.9% NaCl) was used for dilution of the calibration samples.

Table 1.   Comparison of standard and modified assay protocols for von Willebrand factor ristocetin cofactor (VWF:RCo) activity
Assay variableStandard protocolModified protocol
  1. *According to manufacturer, extrapolation to 0.10 IU mL−1 is possible. For concentration > 1.50 IU mL−1 the sample may be diluted with 0.9% NaCl and the result is multiplied by the dilution factor. Combined range based on two calibration curves (high and low). The given platelet count is based on eight different lots with a mean of 1075 × 109 L−1 (SD = 116 × 109 L−1) in each vial that had been reconstituted with 4 mL water. §The final ristocetin concentration was based on the measurement of eight different lots that had been reconstituted with 4 mL H2O where after the platelets were removed by centrifugation and analysis was performed on the supernatant. Mean concentration in reconstituted BC®VW reagent was 0.63 mg mL−1 (SD = 0.05 mg mL−1).

Measuring range0.20–1.50 IU mL−1*0.03–2.00 IU mL−1†
Reagent reconstitutionBC® VW reagent reconstituted with 4 mL H2O, shaken vigorously 2 × 5 sBC® VW reagent reconstituted with 4 mL H2O, shaken vigorously 2 × 5 s, + 3 mL 0.9% NaCl, + 0.2 mL 50 mg mL−1 ristocetin
Diluent for sample and calibration curve0.9% NaClVWFDP
Reaction mixture150 μL reagent, 20 μL 0.9% NaCl, 10 μL plasmaHigh curve: 150 μL reagent, 25 μL VWFDP, 5 μL plasma. Low curve: 150 μL reagent, 30 μL plasma
Final platelet countApproximately 900 × 109 L−1Approximately 500 × 109 L−1
Final ristocetin concentration§0.53 mg mL−11.45 mg mL−1
Limits of detection (LODs)0.10–0.20 IU mL−10.03 IU mL−1

The modified automated protocol (Table 1) differed from the standard protocol in that it included a number of additional features. The first involved introduction of two calibration curves, one with a 5-μL plasma sample volume (high measurement range) and one with an increased plasma sample volume of 30 μL (low measurement range) as described previously [11]. The second modification involved further dilution of the reconstituted BC® VW reagent with 3 mL 0.9% NaCl solution and an increase in the ristocetin content by addition of 0.2 mL 50 mg mL−1 ristocetin to 7 mL of reagent solution. After these modifications, plasma samples and calibration plasma were analyzed in duplicate, and a mean value was used if the deviation between the two results was < 10%. A further modification involved the use of VWFDP as a diluent instead of saline.

Calibration of the VWF:RCo assays was performed automatically by the BCS XP® analyzer using IS 02/150 for VWF in plasma as a reference.

Manual VWF:RCo assay

The manually determined ristocetin activities were used as references. Semi-quantitative determination by visual assessment of platelet agglutination in a dilution series was performed according to the European Pharmacopoeia [16]. One vial of a VW reagent (Siemens Healthcare Diagnostics), containing lyophilized platelets, ristocetin and EDTA, was reconstituted with 2 mL of distilled water immediately before use. In short, the assay involved mixing 50 μL of suitable dilutions of the sample or the reference preparation, using 0.9% NaCl and 5% human albumin as a diluent, with 50 μL VW reagent on a glass slide by moving it gently in circles for 1 min. After the mixture had been allowed to stand for one further minute, the result was read against a dark background with side-lighting. The last dilution that clearly showed visible agglutination indicated the ristocetin cofactor titer of the sample. Diluent was used as a negative control. All samples were analyzed in duplicate. IS 00/514 was used as reference preparation in case of concentrates, whereas for the assessment of plasma samples, the plasma IS 02/150 was used.

VWF:Ag assay

The quantitative immunoassay using the VWF:Ag kit from Siemens Healthcare Diagnostics was performed on the BCS XP® analyzer according to the manufacturer’s recommendations. The assay was calibrated by serial dilution of pooled normal plasma, which was standardized against IS 02/150 for VWF in plasma. The calculated coefficients of variance (CVs) were 2.2% at a level of 1.20 IU mL−1, 3.7% at a level of 0.60 IU mL−1 and 14.3% at a level of 0.10 IU mL−1. The LOD was 0.030 IU mL−1.

Recovery calculations (comparison of standard BCS®, modified BCS® and manual assays)

Four commercially available VWF-containing concentrates [Alphanate® (Grifols Biologicals, Barcelona, Spain), Fandhi® (Grifols), Haemate® (CSL Behring, Marburg, Germany), Wilate® (Octapharma, Vienna, Austria)] and lyophilized control plasmas (N [normal] and P [pathological]) supplied by Siemens Healthcare Diagnostics, and IS 00/514 as a control was used to compare recovery across the different VWF:RCo methods. All products were tested using the manual assay according to the European Pharmacopoeia [16]. This provided the nominal reference value for each concentrate. The assigned values for IS and control plasma were used as the reference values for these standards. Subsequently, recovery of the test products was calculated by dividing the value from the respective automated assay by the manual VWF:RCo value and multiplying by 100.

Validation studies

The validation of the modified automated assay involved accuracy, precision (repeatability and reproducibility) and linearity analyzes using VWFDP spiked with the concentrate IS 00/514 (assigned potency of 9.4 IU mL−1) to concentrations of 1.80, 1.40, 0.90, 0.50, 0.35, 0.20 and 0.05 IU mL−1. For each concentration, five independent dilutions were prepared, aliquoted in three different vials and stored at −70 °C until use. Before analysis, the samples (5 × 7 concentrations = 35 samples) were thawed for 5 min in a 37 °C water bath and carefully mixed before placing in the coagulation analyzer. The protocol was repeated for three independent test runs. At each test run, a new calibration was performed.

Sample stability was followed for a time period of 4 h by leaving the samples at room temperature prior to analysis. Two concentrations were used for the analysis, one high (1.40 IU mL−1) and one low (0.20 IU mL−1). Both samples were prepared by spiking VWFDP with IS 00/514 with five independent dilutions of each concentration. Samples with the same concentrations were chosen for evaluation of the effect of repeated freeze-thawing cycles. One set of five independent dilutions of each concentration was analyzed after 1, 2, 3 or 4 freeze/thaw cycles. The whole procedure was repeated with 30-min freezing cycles. In order to reduce the effect of reagent age on the sample stability tests, new vials of BC® VW reagent were reconstituted every 2 h.

Testing of reagent stability was carried out using reagent that was reconstituted and then kept on-board the recommended position in the cooling area of the analyzer for 6 h. Five sets of five independent dilutions of a single concentration (0.90 IU mL−1) were used for the test, and samples were thawed just prior to analysis.

LOD was measured by two different procedures. One method was based on the regression model of the low calibration curve using the formula: 3.3 × standard error of statistical estimate/slope. The other method was based on the repeated measurement (n = 10) of a VWD type 3 plasma, and calculated by multiplying the SD of the obtained raw value by 3.3. The obtained value was compared with the low calibration curve to determine the corresponding value. For values below the lowest calibration point (0.031 IU mL−1), a LOD < 0.031 IU mL−1 was assumed.

The Clinical and Laboratory Standards Institute protocol (CLSI EP10-A3) for evaluation of quantitative methods was used to compare the effect on analytical precision obtained either by the use of stored calibration curves or by a procedure where the assay was calibrated at each new test run. For this evaluation, three plasma pools with different concentrations (high, low and mid level) were prepared using lyophilized control plasma N and control plasma P (Siemens Healthcare Diagnostics). These were reconstituted according to the manufacturer’s instructions and control plasma N was used for the high concentration (approximately 1.20 IU mL−1). Control plasma P was further diluted 1:1 with VWFDP and used for the low concentration (approximately 0.20 IU mL−1). A mid-level concentration (approximately 0.70 IU mL−1) was prepared by mixing equal parts of the high and low plasma pools. Five sets of plasma samples (five independent test runs), with three samples of each concentration, were prepared and stored at −70 °C until analysis.

Data analysis

The validation of the modified automated assay was based on ICH guideline Q2(R1) and involved evaluation of accuracy, precision (repeatability and reproducibility), linearity (including range), specificity and system suitability testing, LOD and stability.

The evaluation of accuracy and precision was based on a variance analytical model with the concentration as a fixed factor and the day as a random factor. For accuracy, the recovery rate as a percentage served as a target value. Repeatability was evaluated over the CV for the residual error, intermediate precision over the CV for the total variance.

Linearity was established using a least-squares algorithm over recovery vs. target values.

LOD was determined based on the standard deviation of the response and the slope. Stability was evaluated using a trend test according to Neumann [17].

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Comparison of assay protocols

Characteristics of the standard and modified automated protocols are presented in Table 1. Compared with the standard automated protocol, the measuring range in the modified protocol has been widened from 0.2 to 1.5 IU mL−1 to 0.03–2.00 IU mL−1, leading to a lower LOD.

Recovery

The four commercially available VWF-containing concentrates (A, B, C, D) and the IS were used to analyze the assay performance using the standard and modified protocols. When the four concentrates and the IS are compared with a reference value (the values obtained using the manual method for the concentrates and the assigned value for the IS), the standard protocol underestimates the VWF:RCo recoveries of the individual concentrates and the IS, compared with the other assay methods (Fig. 1). This can be compensated for by increasing the ristocetin concentration, as done in the intermediate and final modified protocols. The calculated ratios of VWF:RCo/VWF:Ag can also illustrate that the modified automated test protocol has a functional capacity that is in agreement with the manual assay used to assign the concentrate potencies. Thus, the manual VWF:RCo assay resulted in ratios between 0.75 and 0.90 for the five different concentrates whereas the corresponding ratios for the modified protocol was in the range 0.69–0.89. The calculated ratios based on the standard automated VWF:RCo protocol were lower (0.39–0.58).

image

Figure 1.  Comparison of recoveries between three assay protocols of four commercially available von Willebrand factor (VWF) concentrates (A, B, C, D) and IS 00/514 (assigned potency of 9.4 IU mL−1). The bars represent the standard BCS® protocol (gray), an intermediate adaptation of the protocol (white) and the fully modified assay (black). For the intermediate protocol, 55 μL of 50 mg mL−1 ristocetin was added to the reconstituted BC® VW reagent (without the addition of 3 mL 0.9% NaCl as for the full modification). Thus, the intermediate protocol resulted in less ristocetin (about 75%) than the fully modified protocol but a higher platelet count. The four concentrates were analyzed and compared with IS 00/514.

Download figure to PowerPoint

Stability

To assess the stability of the samples, they were either subjected to up to four repeated freeze/thaw cycles or left at room temperature for up to 4 h before the analyzes with the modified BCS® method (Fig. 2). For both a high and a low dilution, the activity measurements stayed stable for both procedures, demonstrating a high stability of the assay samples. Additionally, the on-board stability of the reagent was assessed over a period of 6 h (Table 2). The mean VWF:RCo activity with the modified BCS® method was approximately 1 IU mL−1 over the whole measuring period, but there was a definite trend for the VWF:RCo to increase from 0.99 IU mL−1 at the beginning to 1.26 IU mL−1 after 6 h on-board.

image

Figure 2.  Evaluation of sample stability. Measurements of von Willebrand factor ristocetin cofactor (VWF:RCo) activity of plasma samples spiked with IS 00/514 VWF concentrate using the modified BCS® method. Black solid lines indicate stability over time at room temperature (h) and gray hatched lines indicate stability after repeated freeze/thaw cycles (up to 4). This is presented for both the lower and upper concentration range of the sample.

Download figure to PowerPoint

Table 2.   On-board reagent stability
Time (h)00.5246
Mean result, IU mL−1 (n = 5)0.991.031.041.141.26

Accuracy and precision

For the modified BCS® protocol, the calibration curve was split into two overlapping parts. In this way, one calibration curve is created with less sample volume for high-range measurements and one curve is created using with increased sample volume for measurements in the low range. Thus, with an increase in sample volume, a LOD of 0.03 IU mL−1 was obtained (Table 1) and the overall dose response was linear between 0.03 and 2.00 IU mL−1 (Fig. 3). As the two curves overlapped in the range 0.25–0.50 IU mL−1, the decision was taken to analyze samples with activities < 0.50 IU mL−1 using the ‘low’ curve protocol.

image

Figure 3.  Investigation of linearity as an illustration of accuracy using the modified method for plasma samples spiked with IS 00/514 von Willebrand factor (VWF) concentrate. The observed mean activity value (IU mL−1) was plotted against reference activity (IU mL−1). The regression line was determined with a correlation coefficient, r, of 0.998.

Download figure to PowerPoint

The calculated mean values resulted in high levels of accuracy with recoveries that are close to the theoretical reference concentrations of the IS as illustrated in Table 3. Overall repeatability was within a CV of 7.5% and 1.3% in the lower and medium concentration ranges, respectively. Reproducibility ranged from a CV of 15.0% for a reference concentration of 0.05 IU mL−1 to 1.5% for a medium reference concentration of 0.90 IU mL−1. Fig. 3 displays the accuracy of the measurements. When plotted against the reference activity of the IS, the observed mean activity resulted in a regression line with a slope close to 1 (0.993) and a correlation coefficient of 0.998, indicating a high accuracy of the measurement.

Table 3.   Results from testing of diluted concentrations of the reference standard (IS) with the modified BCS® protocol
Reference concentration (U mL−1)Mean concentration ± SD (IU mL−1)Mean recovery (%)Overall repeatability (CV, %)Reproducibility (CV, %)
  1. IS was diluted in VWFDP using its assigned potency. A total of 15 samples of each concentration were prepared and tested on 3 days (five samples of each concentration/test day).

1.801.79 ± 0.04992.12.3
1.401.46 ± 0.051042.23.2
0.900.97 ± 0.021081.31.5
0.500.59 ± 0.021183.03.3
0.350.41 ± 0.021173.93.8
0.200.20 ± 0.021004.17.3
0.050.05 ± 0.011007.515.0

To assess the necessity of obtaining new calibration curves for each assay run using the modified BCS® method, the imprecision CV was analyzed comparing stored calibration curves with new curves for each assay run (Table 4). Overall, the total imprecision CV was even smaller with stored calibration curves. Hence, stored calibration curves can be used for this method without loss of precision.

Table 4.   New vs. stored calibration curves and effect on precision
Plasma pool levelTotal imprecision CV (%) (calibration each run)Total imprecision CV (%) (stored calibration)
  1. Plasma pool based on control standards N and P.

High (1.20 IU mL−1)4.43.2
Mid (0.70 IU mL−1)8.86.9
Low (0.20 IU mL−1)5.85.0

Comparison of assays in VWD patient samples

Plasma samples from seven VWD type 2 (six 2A and one 2B) and seven type 3 patients were collected after washout of any treatment. Samples were analyzed with the standard and modified BCS® VWF:RCo methods and VWF:Ag. The individual values obtained for these patients are presented for both methods in Table 5. These data clearly illustrate the increased sensitivity of the modified VWF:RCo assay protocol compared with the standard BCS® protocol. They also indicate that the increased sensitivity critical to type 3 investigations does not come at the cost of falsely estimating VWF functionality in type 2 investigations. Based on the values given in Table 5, the calculated mean VWF:RCo/Ag ratio for the 7 VWD type 2 patients was 0.32 (range 0.12–0.47) for the modified assay protocol whereas the corresponding ratio was 0.56 (range 0.34–0.93) for the standard automated protocol, which further strengthen the clinical capacity of the modified assay protocol. None of the seven patients with VWD type 3 had any detectable antigen (below LOD of the assay that was 0.03 IU mL−1). A similar phenotype was shown in six out of the seven type 3 patients using the modified VWF:RCo assay and only one had a detectable level of 0.054 IU mL−1. The standard BCS® protocol has a LOD that is set at 0.10 IU mL−1 and not suited for VWD type 3 diagnosis. Nevertheless, the standard BCS® protocol yielded levels above the LOD in four out of the seven patients with VWD type 3.

Table 5.   Results of testing patients with von Willebrand factor disease (VWD) (type 2 and type 3 subtype) after washout of any replacement therapy
Patient noVWF:Ag (IU mL−1) LOD = 0.030 IU mL−1Modified BCS® (IU mL−1)LOD = 0.031 IU mL−1Standard BCS® (IU mL−1) LOD = 0.100 IU mL−1
  1. All samples were analyzed as duplicates and the mean results are given in the table. *Both results are shown, as one result was above and one result was below, the assay LOD.

Type 2 VWD (2A or 2 B)
 1 (2A)0.460.200.16
 2 (2A)0.270.070.10
 3 (2A)0.380.090.13
 4 (2B)0.170.020.12
 5 (2A)0.280.080.13
 6 (2A)0.160.070.12
 7 (2A)0.150.070.14
Type 3 VWD
 1< 0.030< 0.0310.103/< 0.100*
 2< 0.030< 0.0310.126
 3< 0.030< 0.031< 0.100
 4< 0.030< 0.031< 0.100
 5< 0.030< 0.031< 0.100
 6< 0.0300.0540.128
 7< 0.030< 0.0310.111

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Automated VWF:RCo assays have the advantage of being easier to perform, requiring small sample volumes, facilitating a high throughput, and providing faster turnaround time to generate assay results. However, the established automated protocols were developed essentially for patient diagnosis in the medium to medium-low VWF:RCo activity range, without optimization for measuring low activity levels in patient diagnostic plasma samples or in plasmas obtained hours after therapeutic VWF replacement.

The conditions for the ristocetin-induced platelet agglutination vary between protocols and the optimal concentration of ristocetin is merely based on empirical grounds. Studies with manual agglutination of formalin-fixed platelets have shown an increase in the rate of platelet agglutination with increasing concentrations of ristocetin if the plasma content is constant [18]. However, it appears that the useful range of ristocetin is only between 1 and 2 mg L−1. At higher concentrations of ristocetin, precipitation of plasma proteins will occur [18,19]. It is also important to note that the reaction conditions of automated assay protocols are different from the manually performed assays in the sense that less plasma and higher platelet count are used in the automated protocols. Furthermore, in manual assays the calculated result represents the maximum rate of agglutination whereas in the automated protocol by Siemens the raw data are the time it takes for a change of 300 mAU. Thus, the optimal ristocetin concentration may be different for different protocols and it is likely that there is a threshold concentration of ristocetin that is needed to sustain optimal sensitivity without the induction of plasma protein precipitation. The manufacturer does not declare the ristocetin concentration of the BC® VW reagent so we measured the content (in the supernatant of reconstituted reagent) in eight different lots that resulted in a mean concentration of 0.63 ± 0.05 mg mL−1. This is a rather low concentration, but may be explained by the fact that some of the ristocetin is actually bound to platelets and is thereby not free in solution. Our modifications, involving the addition of 0.2 mL of a 50 mg mL−1 ristocetin solution to each vial of reconstituted reagent, resulting in a final ristocetin concentration of 1.45 mg mL−1. This concentration is below the threshold for precipitation of proteins [19], which was validated by the absence of an absorbance change during the test reaction under conditions without platelets or without VWF (A. Hillarp, unpublished observations).

In our experience, the original BCS® assay is not linear through the range of concentrations [11]. This phenomenon is circumvented by the use of two separate calibration curves for high and low concentration ranges, which allow the extension of the measuring range, especially on the low end, making this assay better suited for diagnosis of patients with low VWF:RCo activity, for monitoring VWF:RCo and during pharmacokinetic studies. This resulted in a low LOD, without compromising the precision of the assay, making it a notable alternative to other variants of the VWF:RCo assay. We also showed that the calibration curves could be stored without loss of precision (Table 4), which facilitates rapid and individualized sample testing.

In a comparison of a number of VWF/FVIII concentrates, where the VWF:RCo potencies assigned by manual aggregometry were compared with the potency estimates obtained by the standard automated test protocol, we observed that the standard protocol resulted in much lower activities than expected (Fig. 1). With the modifications to the standard method it was possible to obtain better correlation with the manual assay when testing different VWF/FVIII concentrates and with the assigned potency of the IS. Besides our own investigations, there are three other published reports looking at automated protocols based on commercially available lyophilized platelets, and they all differ in platelet count and final ristocetin concentration in the analyte [11,13,14,20]. In our review of these studies, it was clear that the VWF:RCo activity results could be affected by changing these parameters. Our final regent modification involved reconstitution of one vial of BC® VW reagent with 4 mL of distilled water, 3 mL of saline and addition of 200 μL of ristocetin (50 mg mL−1). With this modification, the potency estimates of the tested concentrates and the IS for both plasma and concentrates were close to the assigned potencies of the concentrates using a manual agglutination-based assay and the potency assignment of the standard by the NIBSC.

In testing of patient samples, the modified assay with its improved LOD, showed the ability to discriminate the lower values as seen in type 3 patients. The data also demonstrated that the increased sensitivity critical to type 3 investigations did not come at the cost of falsely estimating VWF functionality in type 2 investigations. These findings together allow for increased accuracy in type 2 and type 3 diagnosis, and determining the true half life characteristics during PK studies, where the longer time points would not be observable with the standard method. Patients with type 2M phenotype were not included in this study. Type 2M is a rare qualitative VWD variant characterized by decreased binding to platelets despite normal VWF multimers. However, the diagnosis of type 2M is problematic with a phenotypic definition that may be difficult to distinguish from the type 1. We might speculate that the increased sensitivity of our modified VWF:RCo assay may provide a predictive avenue for diagnosing type 2M VWD, where functionally accurate assessment of plasma VWF is necessary for diagnosis but this needs to be investigated further.

The precise quantification of very low levels of VWF:RCo activity in individuals with type 3 VWD may be clinically useful and provide predictive genotypic and phenotypic relationships. That is, recessive type 3 VWD appears to be because of over 50 different gene mutations arising randomly throughout the VWF gene, resulting in a null phenotype, characterized by extremely low or undetectable levels of VWF in both platelets and plasma [21]. Traditionally, the diagnosis of type 3 VWD was predicated on the VWF:Ag levels based on sensitive immunoassays, rather than defining the diagnosis on VWF:RCo assay results, because of the limitations of that assay as described above. Recent reports suggest that platelet morphological changes [22], likelihood of alloantibody formation [21] and perhaps clinical and laboratory phenotypes, may be associated with specific VWF genetic haplotypes. The ability to measure low levels of VWF:RCo activity may provide increased sensitivity to correlate VWF structure and function in type 3 patients.

The modifications presented here for the standard automated protocol using the Siemens Healthcare Diagnostics BCS® analyzer are easy to perform and can be easily adapted depending on the purpose of testing. The change of reagent composition is done with commercially available ristocetin and the curve programs are available on the standard analyzer. For diagnosis of patients with VWD and for monitoring of patients treated with VWF/FVIII concentrates, it would be preferable to use the two-curve mode of the protocol for extended measuring range and to perform calibration with standard plasma that adheres to the current IS for VWF in plasma.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

The authors would like to acknowledge the expert technical assistance of Mrs I. Hemborg at the laboratory in Malmö and S. Janisch and C. Zapfl from the research and development team at Octapharma Vienna. Results from manual assays were provided by the laboratory technician team/Quality Control, Octapharma Vienna.

Disclosure of Conflict of Interests

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

A. Hillarp has a consultancy agreement with Octapharma. C. Kessler has served on advisory boards for Octapharma, Grifols and CSL, and has participated in clinical and laboratory research studies sponsored by Octapharma. The other authors state that they have no conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References