The overall performance of the newly developed VWF:CB and OVC VWF:Ag assays was excellent, and the linearity study revealed an optimal range of optical densities (0.23–0.98). Use of the plasma pool relative concentration of 100% in the VWF:Ag assay for the calculation of R2 and slope was often excluded; the dilutions were not adjusted to include this concentration in the optimal absorbance range, in order to take advantage of this range for lower values where accuracy was more clinically imperative. The plasma pool relative concentration of 100% was typically included in the VWF:CB assay due to generally lower absorbances [optical density (OD)] in this assay. The linearity study also revealed poor accuracy of the VWF:Ag and VWF:CB assays in the lower optical density range (< 0.23 and 0.13, respectively). The sensitivity of the assays was thus determined to be limited to the 12.5% dilution corresponding to optical densities of 0.23 and 0.13 as mentioned previously.
The intra-assay CVs were excellent over a wide range of VWF:Ag concentrations with an average of 7.87% for the OVC VWF:Ag assay and 4.4% for the VWF:CB assay. Although previous reports have cited CVs as low as 3.8% for the VWF:Ag assay, the VWF:Ag concentration used for that determination was not stated (3
). Using the assessment of repeatability from the “high” VWF:Ag concentration (85%) our assay performed with a CV of 4.2%, which is comparable to that found in the previous study (3
). In addition, the average CV for the OVC VWF:Ag assay is comparable to a CV of 6.1% reported for the Asserachrom VWF:Ag ELISA assay by the Hemostasis Reference Laboratory. In previous studies, the CV for the VWF:CB has been reported as low as 4.4%, which was consistent with our findings (using the average CV) (3
). Both assays performed well during the evaluation of inter-assay repeatability. Previously reported inter-assay CVs for the VWF:CB have ranged from 1.2% to 11.0% depending on the concentration of VWF present (22
). In our study, the CVs for all VWF:CB concentrations were slightly higher that those of Sabino et al (22
), possibly because of the different time periods used in each study. Sabino et al (22
) used data from assays performed on 14 separate days, whereas the data used to calculate the inter-assay CVs herein were collected over a 10-month period. This approach was chosen because it coincided with sample evaluation, and more accurately reflected the degree of variation throughout our study. However, there are increased numbers of possible confounding factors encountered over such a prolonged period, such as numerous batches of reagents, changes in lots of antibodies, and variable environmental conditions, that may have further contributed to the degree of variation. The inter-assay CV for the OVC VWF:Ag assay (range: 3.8–9.2%) was similar to the 3.0–7.2% range reported for the Asserachrom VWF:Ag ELISA assay.
Agreement of the OVC VWF:Ag assay with the Asserachrom VWF:Ag ELISA assay was almost perfect when assessed using concordance correlation. This statistical test is superior to other types of analyses, such as the paired t
-test and Pearson’s correlation, which only show association between data, not necessarily agreement (20
). The concordance correlation is essentially analogous to Cohen’s weighted kappa and is used as a measure of agreement between 2 clinicians when the response is ordinal (20
). The Bland-Altman plot is used for assessment of within-animal variation, as well as how interchangeable the assays are in a clinical context. Provided differences, within the mean difference (+/− 2 s
) of the difference, are not clinically important, the 2 measurement methods can be used interchangeably (17
). Our plot () showed systematic variation in the differences, that is, increased difference with increased mean. While this pattern often suggests that a logarithmic transformation is necessary, these data responded best visually to a square root transformation. Unfortunately, a log transformation is the only transformation giving back transformed differences that are easy to interpret, and use of any other transformation is not recommended in this context (17
). Thus, nontransformed data showing systematic variation results in limits of agreement that are wider than necessary for small mean VWF:Ag concentrations, and narrower than they should be for larger mean VWF:Ag concentrations (17
). Additional markers, therefore, were inserted to represent the mean (+/− 1 s
) to assist in the visual assessment of clinical relevance of the difference between assays. For the smaller mean VWF:Ag concentrations, most differences were represented by a tight cluster of data within 1 standard deviation of the mean. Although the data were more loosely clustered as the mean VWF:Ag concentration increased, up to a mean concentration of approximately 80%, from 80% to 85% of the data points were within the mean (+/− 1 s
). A difference of 40% between assays is clinically unacceptable; however, this degree of disagreement was only seen consistently in the higher VWF:Ag concentration ranges, where patients are considered VWD negative; the influence on clinical interpretation in this range is minimal. In contrast, the least amount of disagreement, as mentioned, was in the lower mean VWF:Ag concentrations where a high level of agreement is very important. Overall, this graphical assessment of the 2 assays suggested that while they showed excellent agreement through the concordance correlation, strictly speaking, these 2 assays are not interchangeable. This is somewhat expected as test results from different laboratories are generally not directly comparable due to differences in plasma standards, assay techniques, and reference intervals (23
). However, the level of agreement in the range of assay results having the most effect on patient management was likely sufficient to justify using the OVC VWF:Ag assay in place of the Asserachrom VWF:Ag ELISA assay in a clinical context.
Diagnostic testing in humans has found that the decrease in function of VWF parallels the level of VWF:Ag concentration in type 1 and 3 disease, suggesting there should be a high degree of association between these tests (1
). Due to the current lack of a reference assay for comparison with the newly developed VWF:CB assay, the relationship between the concentration and function of VWF was used to assess performance of the VWF:CB assay. The VWF:CB showed a strong association with both the Asserachrom VWF:Ag ELISA assay and the OVC VWF:Ag assay (R2
= 0.86 and 0.82, respectively). Similarly, assessment of the VWF:Ag to VWF:CB ratio in this study yielded expected results for a type 1 VWD population, as the ratio for these patients should generally be ≤ 1 (11
). Based on the known relationship between concentration and function of VWF in type 1 VWD, the VWF:CB performance is excellent; however, further studies are required to confirm that the VWF:CB is capable of detecting the disproportionate decrease in VWF function associated with type 2 disease.
In addition to showing excellent agreement, or strong associations with each other, each assay used in this study was able to successfully differentiate groups of VWD negative and VWD positive patients. This finding reinforces the use of these assays in a clinical context for the identification of VWD.
Reference intervals were determined for each of the newly developed assays. The indeterminate values reflect the overlap of results from VWD positive and negative patients and reinforces the variable presentation of this disease. Although the number of Doberman pinschers used in the reference interval determination (VWD negative = 21, VWD positive = 45) was high and could be considered a bias, they were not excluded from the population. The well-known affiliation between this breed and particular disease suggested that their inclusion more accurately reflected the target population. For the VWF:Ag assay reference intervals, the lower cutoff for the VWD negative population was comparable to literature values. However, the reference interval determined for our indeterminate group was wider, making a lower result necessary to confirm disease. The value of a single sample in an animal with an indeterminate VWF:Ag ELISA result is limited by daily and weekly variation in VWF:Ag concentration, and patients falling within this area of the reference interval should be retested to reveal any trends that may be present (24
). For the VWF:CB reference intervals, the lower cutoff for the VWD negative population was higher than that seen for the VWF:Ag assay, and was reflective of the tendency for VWF:Ag to VWF:CB ratios of < 1 in our study. While good screening efficacy of the VWF:CB has been determined in human laboratories, use of the reported reference intervals for screening purposes should be approached with caution (7
). As mentioned, the tendency of our study population was to have a VWF:Ag to VWF:CB ratio < 1; therefore, the VWF:CB result was typically higher than the corresponding VWF:Ag concentration. However, a VWF:Ag to VWF:CB ratio up to one is quite acceptable in the literature, and with the reported reference ranges a patient determined to be VWD negative based on a VWF:Ag concentration of 80%, and a VWF:Ag to VWF:CB ratio of 1.0 could be inappropriately placed in the indeterminate group if the VWF:CB assay was used alone. As such, based on the study results herein, the VWF:CB appeared to be most valuable when used in conjunction with the VWF:Ag assay.
In conclusion, the overall performance of the newly developed VWF:CB and VWF:Ag assays was excellent. Although, strictly speaking, the OVC VWF:Ag assay cannot be freely interchanged with the Asserachrom VWF:Ag ELISA assay, it is capable of distinguishing VWD positive dogs from VWD negative dogs, and with knowledge of its limitations on either side of the optimal absorbance range, its otherwise excellent performance shows promise for clinical application.