Diagnostic criteria for von Willebrand disease (VWD) differ between guidelines although all require laboratory evidence of low von Willebrand factor (VWF) levels, typically in the setting of a personal bleeding history. The laboratory diagnosis is complicated by interassay and intraassay variability. Further, there are many modifiers of VWF levels that temporarily increase levels from baseline, such as pregnancy, aging, exercise, and oral contraceptives. Iron deficiency anemia is a frequent complication of VWD, and recently has been hypothesized to be enough of a biologic stressor to possibly obscure VWD diagnosis by temporarily increasing VWF levels.¹

In addition, pre-analytic variables such as variations with temperature and time to sample transportation, storage, and preparation with processing of samples can lead to inaccurate VWF assays results.² Our group recently reported significant differences seen between VWF assay results drawn and processed at separate phlebotomy and processing sites (off-site) compared to samples drawn and processed in one location (onsite).³ Normalization of VWF Antigen (VWF: Ag), VWF ristocetin cofactor activity (VWF: RCo) and/or Factor VIII (FVIII) was seen in 40%–60% of patients with abnormal results when testing was repeated with on-site processing under the guidance of a consulting hematologist. Given these challenges, repeat testing at a center with on-site assay processing has been recommended in patients with normal VWF levels and a high index of suspicion.

A subset of patients may not require repeat testing. The utility of repeat testing in patients with initial elevated levels has been evaluated by two different groups. In a study of patients aged 0–18 years, Doshi et al., found that values > 100 IU/dL on VWF Antigen (VWF:Ag) or VWF ristocetin cofactor activity (VWF:RCo) yielded negative predictive values (NPV) of > 95%.⁴ Further, they found that 70% of patients were diagnosed with VWD on their initial testing although all testing was performed at one large academic medical institution. The effect of anemia on VWF levels was not examined although the authors do state that one woman with heavy menstrual bleeding (HMB) in the study was concurrently anemic. Brown et al. performed a similar analysis on adolescent females presenting to the emergency room with acute HMB and found that a VWF:Ag > 100 IU/dL and VWF:RCo > 100 IU/dL had a NPV of 93.2% and 95% respectively.⁵ So, VWF:Ag and VWF:RCo levels were significantly higher at presentation for HMB, but a specific analysis for anemia was not done.

Our group sought to evaluate whether this finding could be generalized to initial VWF assays performed at facilities with offsite processing using data from a retrospective study comparing offsite and onsite testing through 17 institutions across the United States. We also sought to examine the role of anemia in possibly obscuring a VWD diagnosis.

The methods have previously been described in detail.³ Briefly, eligible subjects were females 12–50 years of age who were referred to a hematologist due to concern for a bleeding disorder. All subjects had VWF testing with offsite processing prior to referral, followed by VWF testing with onsite sampling and processing under the supervision of the consulting hematologist. The following data elements were collected: age, referral reason, bleeding symptoms, VWF assays (VWF:Ag, VWF:RCo and factor VIII assay) coagulation and hematologic laboratory results, details of onsite and offsite testing facilities, estrogen use, and final diagnosis as ascertained by the consulting hematologist. Although the most recent guidelines recommend using GP1bM as the platelet binding assay in the diagnosis of VWD, VWF:RCo was used as GP1bM is not widely available in the United States. In the current analysis, we focused on subjects with elevated VWF:Ag and/or VWF:RCo, defined as levels ≥ 100% at the referring (“off-site”) institution. This subset was further classified by the qualifying test: elevated VWF:Ag only, elevated VWF:RCo only, or elevated VWF:Ag and VWF:RCo. Results from the referring institutions were then compared to the initial results from the consulting (“on-site”) institutions. Results from the consulting institutions were classified as low (< 50%), normal (50%–100%), or elevated (≥ 100%). Anemia was defined as a hemoglobin of < 12 g/dL.

A total of 47 subjects, from a cohort of 263 subjects, were identified with elevated VWF:Ag and/or VWF:RCo from the referring institutions. Most of these subjects, 25 (53%), had elevated results for both VWF:Ag and VWF:RCo, while 18 (38%) had elevated VWF:Ag only. Four subjects (9%) had elevated VWF:RCo only. A majority of these subjects (n = 32, 68%) continued to have elevated VWF assays results when repeated at the consulting institution. A third of the subjects had normal laboratory values when repeated at the consulting institution. Five (11%) patients with elevated VWF antigen and/or ristocetin co-factor at the referring institution were eventually diagnosed with VWD, resulting in an negative predictive value (NPV) of 89%. For those patients with elevated VWF: Ag as well as VWF:RCo, the NPV increased to 96%. In those with isolated VWF:RCo or VWF:Ag elevation, NPV was 75% and 83% respectively. Laboratory results collected for the study for these five patients are shown in Table 1.

About half (n = 25, 53%) of those patients with elevated VWF:Ag and/or VWF:RCo levels (at consultation or prior to consultation) were anemic, compared to 18% (n = 39) of those with normal or low levels (chi-square p < 0.001). Three of the five patients (60%) with elevated levels and eventual diagnosis of low VWF or VWD had anemia. Two were severely anemic (hemoglobin < 6) at initial laboratory testing with correction of anemia prior to consultation. Eleven (23%) patients with elevated levels were ultimately diagnosed with a different bleeding disorder.

This study demonstrates a similar, though slightly lower, NPV for VWF:Ag or VWF:RCo levels > 100 IU/dL in the diagnosis of VWD compared to prior reports. The role of off-site processing in these findings is difficult to ascertain as typically pre-analytical variables will result in falsely low VWF levels. One limitation of the data arises from the VWF assays given a wide coefficient of variation, as well as the fact that they were not performed using identical instrumentation and reagents. Interestingly, the proportion of patients with anemia was higher in those patients with elevated VWF levels, adding support to the hypothesis that anemia is a sufficient stressor to increase VWF levels. The lowest NPV was seen in those patients with isolated elevation of VWF:Ag which might be expected given this finding can be seen in patients with Type 2 VWD.⁶ However, this can also be seen with Type 1 VWD and of the five subjects with an eventual diagnosis of VWD, only one was diagnosed with Type 2 VWD. All diagnoses were made prior to the recent VWD diagnosis guidelines which suggest using an activity to antigen ratio of < 0.7 as the cut-off for diagnosis of Type 2 VWD.⁷ Following these recommendations, some patients may have subsequently been reclassified. These findings reinforce prior reports that VWF levels above 100 IU/dL may be used to rule out VWD in a specific subset of patients. However, repeat testing should be considered in patients with a significant history of bleeding, concurrent anemia, or other biologic stressors even in the setting of elevated levels. Since there exists the possibility of other hemostatic disorders as we observed despite a VWF level exceeding 100%, referral to a hematologist for further specialized coagulation testing is warranted.

血管性血友病 (VWD) 的诊断标准因指南而异，尽管都需要实验室证据证明血管性血友病因子 (VWF) 水平低，通常是在个人出血史的情况下。实验室诊断因批间和批内变异而变得复杂。此外，有许多 VWF 水平调节剂会从基线暂时增加水平，例如怀孕、衰老、运动和口服避孕药。缺铁性贫血是 VWD 的常见并发症，最近有人假设它是足够的生物应激源，可能会通过暂时增加 VWF 水平来掩盖 VWD 的诊断。¹

此外，分析前变量（例如随温度和样品运输、储存和制备以及样品处理时间的变化）可能导致 VWF 检测结果不准确。²我们小组最近报告了在单独的采血和处理地点（异地）提取和处理的 VWF 测定结果与在一个地点（现场）提取和处理的样本之间存在显着差异。³VWF 抗原 (VWF: Ag)、VWF 瑞斯托霉素辅因子活性 (VWF: RCo) 和/或因子 VIII (FVIII) 的正常化在 40%–60% 的异常结果患者中出现咨询血液学家的指导。鉴于这些挑战，建议对 VWF 水平正常且高度怀疑的患者在具有现场检测处理的中心进行重复检测。

一部分患者可能不需要重复测试。两个不同的小组评估了在初始水平升高的患者中重复检测的效用。在一项针对 0-18 岁患者的研究中，Doshi 等人发现 VWF 抗原 (VWF:Ag) 或 VWF 瑞斯托菌素辅因子活性 (VWF:RCo) 的值 > 100 IU/dL 会产生阴性预测值 (NPV) > 95%。⁴此外，他们发现 70% 的患者在最初的测试中被诊断出患有 VWD，尽管所有测试都是在一家大型学术医疗机构进行的。贫血对 VWF 水平的影响没有被检查，尽管作者确实指出研究中的一名女性月经过多 (HMB) 同时贫血。布朗等人。对因急性 HMB 到急诊室就诊的青春期女性进行了类似的分析，发现 VWF:Ag > 100 IU/dL 和 VWF:RCo > 100 IU/dL 的 NPV 分别为 93.2% 和 95%。⁵因此，就诊时 HMB 的 VWF:Ag 和 VWF:RCo 水平显着较高，但未对贫血进行具体分析。

我们的小组试图评估这一发现是否可以推广到在具有异地处理的设施中进行的初始 VWF 检测，使用来自一项回顾性研究的数据，该研究比较了美国 17 家机构的异地和现场测试。我们还试图检查贫血在可能掩盖 VWD 诊断方面的作用。

前面已经详细描述了这些方法。³简而言之，符合条件的受试者是 12-50 岁的女性，她们因担心出血性疾病而被转诊至血液学家。所有受试者在转诊前都进行了 VWF 检测并进行了异地处理，然后在咨询血液学家的监督下进行了 VWF 检测并进行了现场采样和处理。收集了以下数据元素：年龄、转诊原因、出血症状、VWF 检测（VWF：Ag、VWF：RCo 和因子 VIII 检测）凝血和血液学实验室结果、现场和非现场检测设施的详细信息、雌激素使用和最终诊断由咨询血液学家确定。尽管最近的指南推荐使用 GP1bM 作为 VWD 诊断中的血小板结合测定，但由于 GP1bM 在美国并未广泛使用，因此使用 VWF:RCo。在目前的分析中，我们专注于 VWF:Ag 和/或 VWF:RCo 升高的受试者，定义为参考（“非现场”）机构的水平 ≥ 100%。该子集通过资格测试进一步分类：仅升高的 VWF:Ag、仅升高的 VWF:RCo，或升高的 VWF:Ag 和 VWF:RCo。然后将参考机构的结果与咨询（“现场”）机构的初始结果进行比较。咨询机构的结果分为低 (< 50%)、正常 (50%–100%) 或升高 (≥ 100%)。贫血定义为血红蛋白 < 12 g/dL。然后将参考机构的结果与咨询（“现场”）机构的初始结果进行比较。咨询机构的结果分为低 (< 50%)、正常 (50%–100%) 或升高 (≥ 100%)。贫血定义为血红蛋白 < 12 g/dL。然后将参考机构的结果与咨询（“现场”）机构的初始结果进行比较。咨询机构的结果分为低 (< 50%)、正常 (50%–100%) 或升高 (≥ 100%)。贫血定义为血红蛋白 < 12 g/dL。

来自 263 名受试者的队列中，共有 47 名受试者被鉴定出来自转诊机构的 VWF:Ag 和/或 VWF:RCo 升高。这些受试者中的大多数（25 名 (53%)）的 VWF:Ag 和 VWF:RCo 结果均升高，而 18 名 (38%) 仅 VWF:Ag 升高。四名受试者 (9%) 仅 VWF:RCo 升高。当在咨询机构重复时，这些受试者中的大多数（n = 32, 68%）继续具有升高的 VWF 检测结果。当在咨询机构重复时，三分之一的受试者具有正常的实验室值。转诊机构中 VWF 抗原和/或瑞斯托霉素辅因子升高的五名 (11%) 患者最终被诊断为 VWD，阴性预测值 (NPV) 为 89%。对于那些 VWF:Ag 和 VWF:RCo 升高的患者，NPV 增加到 96%。在那些孤立的 VWF 中：RCo 或 VWF:Ag 升高，NPV 分别为 75% 和 83%。为这五名患者的研究收集的实验室结果见表 1。

VWF:Ag 和/或 VWF:RCo 水平升高（咨询时或咨询前）的患者中约有一半 (n = 25, 53%) 贫血，而正常或咨询前的患者中有 18% (n = 39)低水平（卡方p  < 0.001）。水平升高并最终诊断为低 VWF 或 VWD 的五名患者中有三名 (60%) 患有贫血。其中两人在初始实验室检测时严重贫血（血红蛋白 < 6），并在咨询前纠正了贫血。11 名 (23%) 水平升高的患者最终被诊断出患有不同的出血性疾病。

该研究表明，与之前的报告相比，VWF:Ag 或 VWF:RCo 水平 > 100 IU/dL 的 NPV 相似，但略低。异地处理在这些发现中的作用很难确定，因为典型的分析前变量会导致 VWF 水平错误地降低。数据的一个限制来自 VWF 分析，因为变异系数很大，而且它们不是使用相同的仪器和试剂进行的。有趣的是，在 VWF 水平升高的患者中，贫血患者的比例更高，这增加了对贫血是增加 VWF 水平的足够压力的假设的支持。在那些 VWF:Ag 单独升高的患者中观察到最低 NPV，鉴于在 2 型 VWD 患者中可以看到这一发现，这可能是预料之中的。⁶然而，这也可以在 1 型 VWD 和最终诊断为 VWD 的五名受试者中看到，只有一名被诊断为 2 型 VWD。所有诊断均在最近的 VWD 诊断指南之前进行，该指南建议使用 < 0.7 的活性抗原比作为诊断 2 型 VWD 的临界值。⁷根据这些建议，一些患者可能随后被重新分类。这些发现强化了先前的报告，即高于 100 IU/dL 的 VWF 水平可用于排除特定患者亚组中的 VWD。然而，即使在水平升高的情况下，对于有明显出血史、并发贫血或其他生物应激源的患者，也应考虑重复检测。正如我们观察到的，尽管 VWF 水平超过 100%，但仍存在其他止血障碍的可能性，因此有必要转诊给血液学家进行进一步的专业凝血测试。