Laboratory diagnosis of von Willebrand disease in the age of the new guidelines: considerations based on geography and resources

von Willebrand disease (VWD) is considered the most common bleeding disorder and arises from deficiency and/or defect in the adhesive plasma protein von Willebrand factor (VWF). Diagnosis of VWD requires clinical assessment and is facilitated by laboratory testing. Several guidelines for VWD diagnosis exist, with the latest American Society of Hematology, International Society on Thrombosis and Haemostasis, National Hemophilia Foundation, and World Federation of Hemophilia 2021 guidelines presenting 11 recommendations, some of which have drawn controversy. In the current narrative review, we provide additional context around difficulties in laboratory diagnosis/exclusion/typing of VWD, with a focus on developing countries/resource-poor settings. In particular, there are many variations in assay methodology, and some methods express high assay variability and poor low-level VWF sensitivity that compromises their utility. Although we favor an initial 4-test assay panel, comprising factor (F) VIII coagulant activity, VWF antigen, VWF glycoprotein Ib binding (VWF:GPIbR or VWF:GPIbM favored over VWF Ristocetin cofactor) and VWF collagen binding, we also provide strategies for laboratories only able to incorporate an initial 3-test assay panel, as favored by the latest guidelines, to improve diagnostic accuracy.


| I N T R O D U C T I O N
von Willebrand disease (VWD) is reportedly the most common congenital bleeding disorder and may also arise as an acquired defect called acquired von Willebrand syndrome (AVWS) [1,2]. VWD and AVWS arise as a result of a deficiency of, or defect in, the adhesive plasma protein called von Willebrand factor (VWF). In addition to plasma, VWF also resides in the alpha granules of platelets and the Weibel Palade bodies of the vascular endothelium.
VWF performs several functions [3], including (i) binding to platelets-primarily via the glycoprotein Ib (GPIb) receptor, but additionally to GPIIb/IIIa (also known as integrin αIIbβ3); (ii) binding to the subendothelial matrix, primarily via the protein collagen; and (iii) binding to coagulation factor (F) VIII (FVIII) and protecting this protein from degradation. The main aim of these functions is to facilitate primary hemostasis and contribute to secondary hemostasis, thus preventing blood loss through injury. In brief, upon tissue injury, VWF binds to the damaged vessel subendothelium (via collagen) and to platelets (via GPIb), thereby anchoring platelets to sites of injury. This process leads to platelet activation, release of granule contents, including more VWF and coagulation proteins (FV and fibrinogen), and eventual platelet aggregation. VWF also delivers FVIII to the injury site, and with platelet released cargo, helps facilitate secondary hemostasis, in part conducted on the activated platelet surface, permitting conversion of fibrinogen to insoluble fibrin, and formation of stable platelet plugs.
Fundamentally, failures of VWF to bind to platelets (via GPIb), collagen, or FVIII, compromises its function, and may lead to VWD/ AVWS and bleeding [1,2]. In the hemostasis laboratory, various tests are performed to assess VWF level and function ("activity") [4]. These in vitro tests try to "mimic" what happens in vivo, albeit not exactly matching in vivo interactions. The aim of this narrative review is to discuss diagnosis or exclusion of VWD/AVWS from the laboratory perspective. We explain the tests used by laboratories to identify or exclude VWD, how this process is facilitated using test panels, and how reflexing to additional tests helps to classify (or type) patients with VWD. We discuss this in view of available VWD diagnosis guidelines, as well as considering geographic realities of resource-poor settings. We therefore aim to provide strategies to circumvent reliance on particular assay(s). Discussion of VWD will in general apply to AVWS.

| C L A S S I F I C A T I O N O F V W D
The classification of VWD remains essentially as outlined in guidance from the International Society on Thrombosis and Haemostasis (ISTH) Scientific Standardisation Committee (SSC) on VWF/VWD [5]. There are 6 main types of VWD: (i) type 1, in which patients present with reduced levels of functionally normal VWF; this now includes type 1C, which is characterized by an increase in VWF clearance from circulation; (ii) type 2 VWD, in which patients present with "dysfunctional" VWF, with or without reduction in plasma levels of VWF, with 4 separate types: (a) type 2A VWD, reflecting reduction of highmolecular-weight multimers (HMWM) of VWF; (b) type 2B VWD, representing hyperadhesive (or gain-of-function) VWF-here, VWF may "spontaneously" bind to platelet GPIb, leading to clearance of both (HMWM) VWF and platelets from circulation (ie, lower residual VWF activity and [mild] thrombocytopenia); (c) type 2N VWD, reflecting loss of VWF FVIII binding (VWF:FVIIIB) activity (thereby, leading to lower plasma FVIII:C levels due to increased clearance); (d) type 2M VWD, reflecting loss of VWF activity not associated with loss of HMWM VWF; and finally (iii) type 3 VWD, reflecting (virtual) absence of plasma VWF. Although not really VWD, since defects lie in the platelet GPIb receptor, inclusion of "platelet type" [PT] or pseudo-VWD in discussions related to testing is helpful, since patients present phenotypically similar to those with type 2B VWD (ie, gain-of-function GPIb leads to "spontaneous" binding to normal plasma VWF, which may lead to clearance of both (HMWM) VWF and platelets from circulation (ie, lower residual VWF activity and [mild] thrombocytopenia) [6]. These gain-of-function concepts are also important for conceptually understanding newer VWF activity assays.
Recognition of different types of VWD is more than academic since patient treatment/management differs according to VWD type, of course in addition to other considerations (eg, type of planned surgery) [7,8]. In particular, desmopressin (DDAVP), a nontransfusional form of therapy, can be utilized for minor surgical cover or to prevent bleeding episodes in type 1 VWD, in particular those with mild disease. Otherwise, the main treatment for VWD is replacement of missing/defective VWF (and in some cases replacement of missing FVIII), using VWF (or FVIII) concentrates. The utility of DDAVP is limited in patients with type 2 VWD, not useful in type 3 VWD, and considered to be contraindicated in type 2B VWD. Utility of DDAVP reflects its ability to stimulate release of VWF already stored in endothelial cells, thus leading to a doubling (or more) of functionally active plasma VWF in type 1 VWD (thereby, explaining its utility here), whereas release of "dysfunctional" VWF is the consequence in type 2 VWD (thereby, explaining general lack of utility here). As no release of VWF is achieved in type 3 VWD, DDAVP is not useful in type 3 VWD. In type 2B VWD, release of gain-of-function (hyperadhesive) VWF may lead to aggravation of thrombocytopenia.
A special consideration is made in type 2N VWD. Here, the main presenting plasma "defect" is a loss of FVIII:C, but this is caused by an inability of VWF to bind and protect FVIII from degradation. Thus, DDAVP will act to release stored dysfunctional VWF, and the low relative FVIII:C will remain. Treatment of 2N VWD is primarily VWF replacement, not FVIII replacement, since providing FVIII will only increase plasma FVIII levels temporarily -infused FVIII will quickly degrade and disappear from circulation. Instead, replaced VWF will bind to and protect any FVIII produced by the patient. Therefore, 2N VWD, reflecting low FVIII:C, needs to be distinguished from hemophilia A (also reflecting low FVIII:C), so that the correct therapy is applied (VWF replacement in 2N VWD; FVIII replacement in hemophilia A) [2,7].
This depends on how VWD is defined. Based on epidemiologic considerations, the incidence of VWD can be identified as being around 1% of the general population [1,9]. If based on laboratory testing, with "abnormal" VWF test findings flagged to infer VWD, then the incidence of VWD might be perceived as high as 2% of the general population [10]. This is because reference ranges are defined statistically, based on distribution of test results from healthy populations, either reflecting the mean ± 2× SD of a normally distributed (Gaussian) dataset or based on percentiles for non-Gaussian distributions. In either case, ranges only identify a proportion of the normal population (eg, 95% for mean ± 2SD) as being "normal"; a proportion of the population, including clinically asymptomatic individuals, will thus be flagged as outside this normal range (ie, "abnormal"). A proportion of this "abnormal" group will be above the range (ie, identified as "high"), and a proportion below the range (ie, "low"). If clinicians interpret low levels of VWF or VWF activity as "identifying" VWD, then the potential incidence of VWD may be perceived as being as high as 2% of the general population. Finally, if prevalence of VWD is based on patient numbers attending clinics for investigation or treatment of bleeding or bruising, as then found to have a low level of VWF and/or activity consistent with VWD, this would approximate around 1 in 10,000 of the population (or 0.01%) for developed countries [1]. However, in developing or resource-poor countries, only those with severe disease (or symptoms) would present for investigation or treatment, and prevalence may be identified as much lower than 0.01% [1].
It is also useful to compare perceived prevalence of VWD to hemophilia A, a secondary hemostasis disorder reflecting a loss of FVIII [11,12]. Again, if based on laboratory testing alone, perceived incidence would be similar to VWD, as based on test reference ranges and laboratory/clinical interpretation [10]. If based on patients attending clinics for investigation or treatment of bleeding or bruising, as then found to have low levels of FVIII activity consistent with hemophilia A, this would approximate around 1 in 5000 of the population (or 0.02%) for developed countries [12,13], with most patients being male (given that the affected gene is the X-chromosome). Again, perceived incidence of hemophilia in resource-poor countries may be less, since only those with severe disease are likely to present for investigation/treatment [13].

| L A B O R A T O R Y T E S T S U S E D T O D I A G N O S E V W D A N D E N S U R E A C C U R A T E C L A S S I F I C A T I O N O F V W D T Y P E
These are summarized in Table 1. We believe it mandatory to perform ≥3 different tests as a minimal laboratory panel before VWD can be effectively diagnosed or excluded. These are the FVIII:C assay, a VWF "antigen" (VWF:Ag) assay, and a VWF GPIb binding (VWF:GPIbB) assay.
For the last assay "class," there are now 3 possible options: (a) the historical VWF activity assay, Ristocetin cofactor (VWF:RCo), or more modern alternatives of (b) ristocetin-based assays using recombinant GPIb (VWF:GPIbR), or (c) non-ristocetin based assays using gain-offunction (mutant) (recombinant) GPIb (VWF:GPIbM) [4,14,15]. Such 3-test panels are recommended by the latest American Society of Hematology, International Society on Thrombosis and Haemostasis, National Hemophilia Foundation, and World Federation of Hemophilia 2021 guidelines [15]. In our laboratory, however, and as supported by VWD diagnosis guidelines from the United Kingdom Haemophilia Doctors Organisation, as approved by the British Committee for Standards in Haematology [16], we add a fourth assay (the collagen binding (VWF:CB) assay). Subsequently, additional assays (Table 1) are required to specifically classify patients with type 2 VWD. All of these assays have limitations [4]. Some assays are particularly problematic, yielding high assay variation and poor low VWF level sensitivity [17,18]. The more versions of an assay available, the more variation will be observed between sites using different versions of that assay. This is particularly true for both VWF:GPIbB and VWF:CB assays [17,19].

| The basic 3-test panel for diagnosis/exclusion of VWD
(a) FVIII:C is a mandatory test for VWD since VWF normally binds to and protects FVIII; thus, low levels of VWF are associated with low levels of FVIII [4]. Moreover, low levels of FVIII combined with low levels of VWF compounds bleeding risk (representing defects in both primary and secondary hemostasis). Notably, FVIII is additionally lowered in type 2N VWD. However, FVIII:C testing cannot be used in isolation; a normal level of FVIII does not always exclude VWD (FVIII:C levels will be normal in many patients with "mild" type 1 VWD and also in some patients with type 2B and 2M VWD), and an abnormal FVIII:C does not always establish a diagnosis of VWD (hemophilia A is actually more likely). So, additional tests for VWF level and activity are mandatory.
(b) VWF:Ag is a mandatory test for VWD since it quantifies the level of VWF present in plasma [4]. The lower the VWF level, the greater the bleeding risk. However, VWF:Ag identifies both functional and non-functional VWF forms. Thus, VWF:Ag cannot be used in isolation since normal VWF:Ag levels do not always exclude VWD (levels will be normal in some type 2B and 2M VWD patients), and abnormal VWF:Ag levels, although potentially consistent with VWD, do not identify VWD type (type 1 or type 2 VWD?). Thus, testing for VWF activity is also required.
(c) VWF:GPIbB assays are also mandatory tests for VWD diagnosis/ exclusion since they provide markers for a major VWF activity, being binding of VWF to its platelet receptor (GPIb) [4]. The main question here is which VWF:GPIbB assay to use-VWF:RCo, VWF:GPIbR, or VWF:GPIbM? There are advantages and limitations for each.  I G U R E 1 Geographic disparities in the diagnosis of bleeding disorders, including von Willebrand disease (VWD) (A) Geographic disparities in VWD type diagnosis. The distribution of VWD cases diagnosed is similar among developed countries, and primarily represented by type 1 VWD (>70% of VWD cases), then type 2 VWD (20% of VWD cases), followed by type 3 VWD (<5% of VWD cases). This reflects the "typical" distribution of VWD types. In developing/resource-poor countries, type 3 VWD often represents the majority of VWD cases identified, followed by type 2 and type 1 VWD cases. This probably reflects the "severity" distribution of VWD types, with only severe bleeding presentations requesting treatment. In addition, consanguinity may lead to higher incidence of type 3 in these countries. Data from reference [1]. (B) Geographic disparities in bleeding disorder reporting. In developed countries, most of the estimated burden of bleeding disorder patients are reported to the World Federation for Hemophilia (WFH), with Australia and Canada for example reporting 100% of the estimated number of patients with bleeding disorders. This infers that 100% of the estimated number of patients with bleeding disorders are being "diagnosed" in these countries. In contrast, in developing/resource-poor countries, much fewer patients than estimated are reported; this infers fewer patients with bleeding disorders are being "diagnosed" (or a relative underdiagnosis is occurring). Data from WFH Annual Global Survey 2021 (https://wfh.org/usa/ research-and-data-collection/annual-global-survey/). (C) Geographic disparity in hemophilia vs VWD diagnosis. In line with the expected "similar" prevalence of hemophilia vs VWD, developed countries report similar numbers of cases of hemophilia and VWD to the WFH. In contrast, in developing/resource -poor countries, much higher numbers of persons with hemophilia are reported than with VWD. This infers a relative underreporting (or underdiagnosis) of VWD. Data from WFH Annual Global Survey 2021 (https://wfh.org/usa/research-and-data-collection/ annual-global-survey/). (D) Geographic disparity in hemophilia vs VWD diagnosis part 2. Data from Figure C shown as number of cases reported to the WFH per million of population (left y-axis) for hemophilia (green bars) and VWD (blue bars), and as a ratio of hemophilia/VWD (right y-axis; red bars). In developed countries, similar numbers of cases are reported, with 50 to 100 cases per million (or 1/10,000 cases), and with a corresponding ratio of hemophilia/VWD approximating unity. In contrast, in developing/resource-poor countries, fewer cases of hemophilia are reported; however, much fewer cases of VWD are reported, yielding a corresponding ratio of hemophilia/VWD sometimes in excess of 100. This infers that hemophilia is underdiagnosed, and VWD grossly underdiagnosed, in developing/resource-poor countries. VWD, von Willebrand disease; WFH, World Federation for Hemophilia. ristocetin binding, with the possibility of false VWD diagnosis and incorrect type 2A or 2M VWD assignment (depending on other tests also performed) [20,21]. However, for resourcepoor settings, VWF:RCo has the advantage of being wellestablished and is likely the cheapest of the VWF:GPIbB assays to perform.
(ii) VWF:GPIbR assays represent more modern alternatives to VWF:RCo, primarily performed either by latex agglutination (automated hemostasis analyzer) or chemiluminescence (AcuStar instrument) [4,14,19,22]. In VWF:GPIbR assays, the platelets otherwise used in VWF:RCo assays are replaced with either latex (agglutination assay) or magnetic particles (chemiluminescence assay), and native GPIb (present on platelets for VWF:RCo) replaced with recombinant GPIb as attached to either latex or magnetic particles. VWF:GPIbR assays, being performed on automated platforms, should have lower assay variability and better low VWF level sensitivity than VWF:RCo assays, historically performed on platelet aggregometers, and thus should provide more robust technologies for VWD diagnosis/type assignment. In theory, VWF:GPIbR assays might also be affected by VWF polymorphisms affecting ristocetin binding in VWF:RCo, but several studies show this is not the case (at least for some polymorphisms) [23,24]. In particular, Boender et al. [24] showed similar findings for VWF:GPIbR (assessed by chemiluminescence on AcuStar) and VWF:GPIbM (using the commercial assay on Sysmex CS-5100 analyzer) in 47 patients with the polymorphism p.Asp1472His, with reported values higher than those of VWF:RCo (also performed on the CS-5100). Why the chemiluminescence VWF:GPIbR assay (marketed by the manufacturer as a VWF:RCo) is less sensitive to these polymorphisms than classical VWF:RCo is not known. However, these assays utilize different reagents and are per-  press both defective GPIb and collagen binding), and are reported to have a more severe bleeding phenotype than patients with 2M VWD (who often reflect a loss of GPIb binding, but potentially normal collagen binding, and who do not suffer from loss of HMW VWF) [29][30][31][32][33]. Moreover, when directly compared in surveys of test practice, 3-test panels are associated with "laboratory VWD diagnosis" error rates approximately 2-fold higher than those performing 4-test panels ( Figure 3) [17,19]. This is partly because the fourth test, being the VWF:CB, provides additional diagnostic information, which helps overcome some of the inherent limitations of VWF:GPIbB assays when used alone as the VWF activity assay. For example, laboratories that only perform 3-test panels will never accurately diagnose/discriminate 2A from 2M VWD, and will also have difficulty accurately discriminating type 1 vs type 2 VWD, especially if high assay variability of VWF:GPIbB assays translates to false functional discordance in type 1 VWD, or false functional concordance in type 2 VWD ( Figure 2) [17,19].

| Use of assay ratios
Incorporation of test ratios provides context around specific VWF activity for various assay markers [4]. The main assay ratios are as follows: (a) FVIII:C/VWF:Ag ratio provides context around the specific activity  [18,19]. CLIA, chemiluminescence (immuno)assay; CV, coefficient of variation; HMWM, high molecular weight multimers. would be expected to be concordant (noting, however, that some assays cannot detect levels of VWF to <5 or <10 U/dL; Figure 2F).
These ratios will expectedly be low in types 2A, 2B, and most cases of 2M VWD. The low ratio in these cases is either due to low relative HMWM VWF (2A and 2B VWD) and/or presence of VWF variants expressing defective GPIb binding (2A or 2M VWD).
(c) VWF:CB/Ag ratio provides context around the specific VWF activity of collagen binding, and will be normal (ie, >0.7) in healthy individuals and in those with types 1 and 2N VWD. Ratios are not calculated in type 3 VWD but levels would be expected to be concordant (noting again limits of assay detection; Figure 2F).
The ratio will expectedly be low in type 2A, 2B, and some cases of 2M VWD. The low ratio in these cases is either due to low relative HMWM VWF (2A and 2B VWD) and/or VWF variants expressing defective collagen binding (a proportion of type 2A and 2M VWD).
The ability of the VWF:CB to add value to that of the VWF:GPIbB assays is thus manyfold. First, given high assay variability for most VWF:GPIB assays ( Figure 2)  T A B L E 2 Preanalytical issues affecting the accurate diagnosis of von Willebrand disease (VWD).

Condition or event Effect on VWD diagnosis Comments/strategies
Underfilled blood collection tubes, or pooling of underfilled tubes.
Citrate anticoagulant dilution effect; can lead to false type 1 VWD.
Education of collection and medical staff.
Clotted blood collection or serum. Preferential entrapment of high-molecularweight multimers of VWF (HMWM VWF) that may lead to false type 2A or 2B pattern.
Education of collection and medical staff.
Delayed transport or high temperatures during transport.
Loss of FVIII (highly labile); can lead to false type 2N VWD or hemophilia A pattern.
Education of transport teams.
Transport of whole blood refrigerated or on ice.
Activation of platelets and FVII, leading to potential absorption of HMWM VWF; can lead to false type 2A or 2B pattern.
Education of transport teams.
Plasma filtration (historical process for sample preparation for lupus anticoagulant [LA]). If for investigation of prolonged aPTT, LA may be co-ordered with VWF assays.
Can lead to adhesion of HMWM VWF onto filter.
Education of sample processing team; double centrifugation is now the recommended process for LA sample processing. without appropriate clinical history, can be assigned to a group of "low VWF as a risk factor for bleeding," but not to VWD status.
Another reason type 1 VWD may be diagnosed erroneously in individuals with low, but concordant, VWF levels is because of false concordance due to assay variability ( Figure 2) [17,19]. This is particularly problematic with classical VWF:RCo, but can also occur with VWF:GPIbR and VWF:GPIbM. It can also represent a problem with ELISA based VWF:CB assays. The main recommendation is to repeat VWF tests for confirmation, using fresh samples, to ensure the same pattern is observed. Importantly, the risk of false concordance in type 2 VWD when using both classes of activity assay (ie, VWF:GPIbB and VWF:CB) at the same testing occasion is low, further justifying use of an initial 4-test panel.
(iii) All VWF test results are very low (ie, <5U/dL) but concordant: the patient may have type 3 VWD. However, be warned that some tests have very poor low-level VWF sensitivity and cannot accurately identify levels of VWF <10 U/dL ( Figure 2E). We recommend repeat testing on fresh samples for confirmation, ensuring that tests accurately detect VWF levels <5U/dL before identifying patients as type 3 VWD.
(iv) a low FVIII:C/VWF:Ag is observed (ie, <0.7): the patient may have hemophilia A or 2N VWD, or the low FVIII:C reflects a preanalytical issue (eg, delayed, inappropriate transport; Table 2).
We recommend repeat testing on fresh samples for confirmation, ensuring appropriate collection, storage, and transportation of samples. If test findings are confirmed, hemophilia A and 2N VWD can be differentiated using either a VWF:FVIIIB assay or by genetic analysis of F8 and VWF genes, respectively [15,36].
(v) the VWF:GPIbB/Ag (ie, VWF:RCo/Ag, VWF:GPIbR/Ag, or VWF:GPIbM/Ag) ratio is low or discordant (ie, <0.7): type 2A, 2B, or 2M is possible (as is PT-VWD). Caveats here include false discordance due to assay variability, with VWF:RCo being most problematic. We recommend repeat testing on fresh samples for confirmation. Again, a 4-test panel is better since the risk of a false discordance using both separate assay classes (ie, VWF:GPIbB and VWF:CB) at the same time is low. If discordance is confirmed, then patients should be further evaluated to ensure correct VWD typing. We perform ristocetininduced platelet agglutination (RIPA) first, as this will help identify or exclude 2B or PT VWD (both show response to low ristocetin concentrations) [32,37]. If type 2B or PT VWD is identified, these can be distinguished using RIPA mixing assays or genetic analysis of VWF and platelet GP1b genes, respectively [15,37]

| P R E A N A L Y T I C A L I S S U E S I N V W D D I A G N O S I S / E X C L U S I O N
Laboratories perform sample testing and do not "diagnose" VWD on their own, which involves clinical judgment. Repeat tests for confirmation using a fresh blood sample, and if results discrepant, repeat on a third sample.
Test costs and limited expertise likely to be major barriers to provision of diagnostic services.
Ensure staff are educated in correct interpretation of test results.
FVIII:C testing often performed in isolation (ie, without VWF test panel). Assist partner clinicians to request the right tests for the right patient.
Ensure assays have good low VWF level sensitivity (<5 U/dL of VWF).
Enroll in a good external quality assessment VWF program.
Check validity of the activity/Ag cutoff value for assays you perform; is 0.7 best? 0.7 will capture more type 2 VWD cases, but also some type 1 VWD cases, so need a strategy to undo potential misdiagnosis (eg, repeat testing, multimer analysis). c) The guidelines that are followed: laboratories should follow the latest evidence-based guidelines as much as possible, or else justify any deviations from that guidance. Table 3 provides a synopsis of the most recent VWD diagnosis guidelines, and certain variations related to laboratory testing. Historical context is important, with recommendations to perform VWF:RCo assays in older guidelines "modernized" in latest VWD guidelines to preferential testing with either VWF:GPIbR or VWF:GPIbM (given the theoretically lower assay variability and better low VWF level sensitivity), with VWF:GPIbM "favored" since this should not yield false low values in patients with VWF mutations affecting ristocetin binding [15]. However, we have raised issues with this "suggestion," based on a low grade of evidence, since VWF:GPIbR may also be less affected than VWF:RCo [23,24]. Moreover, in local experience, VWF:GPIbR expresses lower assay variability and better low VWF level sensitivity than VWF:GPIbM [18,19,40]. Another "suggestion" [15] (Supplementary Table 1) is to use a cutoff value of 0.7 instead of 0.5 for VWF activity/Ag ratios to distinguish type 1 and 2 VWD, since a cutoff value of 0.5 may miss some type 2 VWD cases.

| V W D D I A G N O S I S G U I D E L I N E S
Although we agree 0.7 is preferred over 0.5, we instead use a cutoff value of 0.6, which works better with our own instrumentation (AcuStar) and VWF methods (based on chemiluminescence)-indeed, this cutoff (0.6) for this combination permitted complete separation of type 1 and 2 VWD cases assessed by participants of our local external quality assurance program, and outperforms any cutoffs using other methods/combinations ( Figure 2) [19,39,40]. The cutoff value of 0.6 is also recommended by the United Kingdom guidelines [16]. Finally, we utilize an initial 4-test panel instead of a 3-test panel for diagnosis/ exclusion of VWD, again in line with the United Kingdom guidelines [16] but not supported by the latest guidelines [15]. It is clear to us that use of an initial 4-test panel provides for increased assurance around diagnostic accuracy and is also associated with fewer diagnostic errors [17,19].

| A D D I T I O N A L C O N S I D E R A T I O N S F O R R E S O U R C E -P O O R S E T T I N G S
It should be clear from our discourse that accurate identification and diagnosis of VWD is particularly problematic in resource-poor countries ( Figure 1). This may be due to several factors as follows: laboratories in these countries are more likely to perform inexpensive (inhouse or laboratory-developed) tests, smaller test panels (in our experience via the Royal College of Pathologists of Australasia Quality Assurance Program, some laboratories only perform 2-test panels of FVIII:C and VWF:Ag; these laboratories will never be able to accurately diagnose/exclude VWD), may not use the VWF:CB, nor have available multimer or genetic testing [16,17]. In these locations, some additional strategies may be required to permit effective diagnosis/ exclusion of VWD (Table 4).

| C O N C L U S I O N
In this narrative review, we provide suggestions to improve accuracy in laboratory diagnosis of VWD, in part reflecting on geographic aspects and resource-poor countries. We also provide our own algorithmic approach to the diagnosis of VWD, as based on an initial 4-test panel, as well as a potential alternative algorithm based on the more standard initial 3-test panel (Figure 4).

ACKNOWLEDGMENTS
The views expressed herein are those of the authors and are not necessarily those of NSW Health Pathology or other affiliated institutions. We thank staff from the RCPAQAP (Elysse Dean, Sandya Arunachalam) for providing the original data used for some data analysis.

FUNDING
This review did not receive any funding. The views expressed are those of the authors, and not necessarily those of NSW Health Pathology or other affiliated institutions.

AUTHOR CONTRIBUTIONS
E.J.F. conceived the idea for the review and wrote the original draft.