Introduction
Acquired von Willebrand syndrome (AVWS) is a rare and frequently underdiagnosed bleeding
disorder, mainly due to the broad spectrum of possible clinical and laboratory features
affiliated with this condition.[1 ] The mechanisms behind von Willebrand factor (VWF) abnormalities depend upon the
type of underlying disorder and may include increased clearance; enhanced shear stress
and subsequent proteolysis; inhibition of VWF functions; adsorption to the platelet
surface; or, rarely, decreased synthesis.[1 ]
The definition of AVWS was published by the VWF subcommittee in 2000.[2 ] A diagnosis of AVWS can be made based on the following criteria: the existence of
a lack of previous lifelong bleeding incidents and relevant family history, clinical
picture, and laboratory investigation results,[3 ] for example, VWF levels and factor VIII (FVIII) coagulant activity (FVIII:C) are
sometimes decreased, a reduced VWF function/antigen ratio can indicate the existence
of functional disorders, even if the absolute activity is within the normal limit,
a loss or decrease in high-molecular weight (HMW) multimers may also be observable.
The prevalence of AVWS remains unknown and[4 ] the evaluation and management of affected patients may be complex due to the need
for multiple laboratory assays, especially in those in whom the underlying disease
(e.g., prosthetic heart valve or essential thrombocythemia [ET]) necessitates antithrombotic
therapy. The initial laboratory tests used to assess AVWS include VWF level, VWF activity,
and FVIII activity assays. Further tests include VWF multimer analysis, which is a
sensitive tool able to detect the structural abnormalities of VWF even in the context
of normal VWF activity levels. The frequency of the detection of inhibitors, that
is, antibodies against VWF, is low in AVWS. Before 2016, it was not possible to confirm
a suspicion of AVWS in Estonia because of a limitation of available laboratory VWF
assays, while, since 2016, all VWF-related screening assays have been available to
clinicians[5 ] and, recently, a semiautomated VWF multimer assay has been incorporated into routine
clinical practice at the North Estonia Medical Centre (NEMC).[6 ]
[7 ]
Here, we describe the clinical and laboratory data of seven patients diagnosed with
AVWS at NEMC.
Materials and Methods
Patients
We included all consequent patients referred to and assessed at NEMC from the January
1, 2016, to December 31, 2017, who met the criteria for an AVWS diagnosis based on
laboratory findings and bleeding symptoms together with the absence of any previous
history of a bleeding disorder.[3 ]
The most common clinical symptoms were easy bruising, epistaxis, menorrhagia, and
bleeding complications after tooth extraction. The mean age of the patients was 57.4
years (range: 22–80 years). The study group included five women and two men with various
underlying diseases such as non-Hodgkin’s lymphoma (NHL), monoclonal gammopathy of
undetermined significance (MGUS), ET, polycythemia vera (PV), secondary polycythemia
due to cardiovascular diseases, obstructive sleep apnea syndrome, and autoimmune thyroiditis.
All cases were discussed at interdisciplinary meetings between laboratory and clinical
staff. This retrospective study was performed as a collaboration between NEMC and
Helsinki University Hospital, HUSLAB laboratory services, Coagulation Disorders Unit
in partnership with The Twinning Program of the World Federation of Hemophilia (WFH).
The study was performed according to the Declaration of Helsinki and was approved
by the Tallinn Medical Research Ethics Committee.
Blood Sampling
During this study, peripheral venous blood specimens were collected into K2-EDTA tubes
(BD Vacutainer; BD Diagnostics, Plymouth, UK) for a complete blood count, 3.2% sodium
citrate tubes (BD Vacutainer; BD Diagnostics) for coagulation assays, and hirudin
blood tubes (Roche Diagnostics, Switzerland) for platelet aggregation evaluation.
Laboratory Investigations
Based on the laboratory assays available in Estonia, the diagnostic algorithm for
von Willebrand disease (VWD)/syndrome was adopted in this study.[8 ] Initial laboratory evaluations included complete blood count (Sysmex XE-5000; Roche
Diagnostics); prothrombin time (PT) (Neoplastine Cl Plus; Diagnostica Stago, Asnières-sur-Seine,
France); partial thromboplastin time (APTT) (PTT-A; Diagnostica Stago), VWF antigen
(VWF:Ag) (Liatest-VWF:Ag; Diagnostica Stago); FVIII:C determined by a one-stage, clot-based
assay (Diagnostica Stago, France); and VWF activity measured as VWF binding to the
glycoprotein Ib (GPIb) receptor on the platelet surface (VWF:GPIbM) (Innovance VWF
Ac kit; Siemens Healthcare Diagnostics, Marburg, Germany). All parameters were measured
on the STA-R Evolution analyzer (Diagnostica Stago) using commercial kits.
Mixing studies were conducted to determine the etiology of prolonged APTT; the APTT
test was repeated on a mixture of the patient's plasma with normal plasma immediately
and after incubation for two hours at 37°C. Depending on correction, FVIII, FIX, FXI,
FXII, or lupus anticoagulant tests were performed.
Platelet aggregation was measured in whole blood by an impedance multiplate aggregometer
(Roche Diagnostics) using the RISTOhigh test (final concentration of ristocetin: 0.77
mg/mL) and RISTOlow test (final concentration of ristocetin: 0.2 mg /mL). For both,
the measurements were performed within 180 minutes after venipuncture.
The multimeric pattern of VWF was evaluated using the new Hydragel 5 von Willebrand
multimers assay (Sebia, Lisses, France).[6 ]
[9 ]
[10 ]
[11 ] The detailed protocol has previously been described.[12 ] In May 2019, the VWF multimer analysis with 5VWF was accredited in the NEMC laboratory
according to the ISO15189:2012 standard. Both the visual evaluation of the gels and
densitometric analysis were performed. VWF multimers were classified as low-molecular
weight, intermediate-molecular weight, or HMW multimers with densitometry.
Case Series
The main characteristics of the study participants are shown in [Table 1 ]. All patients had other bleeding episodes and no family history for bleeding disorders.
The International Society on Thrombosis and Hemostasis–Bleeding Assessment Tool was
used to score the risk of bleeding (data not presented).
Table 1
Demographic and laboratory characteristics of the study participants
Reference ranges
Case 1
Case 2
Case 3
Case 4
Case 5
Case 6
Case 7
Abbreviations: HMWM, high-molecular-weight multimers; IMWM, intermediate-molecular-weight
multimers; LMWM, low-molecular-weight multimers; NA, nonapplicable; ND, not determined;
VWF: Ag, von Willebrand factor antigen; VWF: GPIbM, VWF activity assays using recombinant
gain-of-function mutant GPIb fragments allowing for the spontaneous binding of VWF
to the mutant GPIb without ristocetin.
Diagnosis
Non-Hodgkin’s lymphoma
ET, JAK2 (V617F)
ET, JAK2 (V617F)
PV
MGUS
Secondary erythrocytosis due to cardiovascular diseases and obstructive sleep apnea
syndrome
Autoimmune thyroiditis
Clinical symptoms
Epistaxis, bleeding complications after tooth extraction
Menorrhagia
Bleeding complications after tooth extraction
Bleeding complications after tooth extraction
Epistaxis
Epistaxis
Spontaneous hematoma
Age, gender
67 F
33 F
61 F
60 F
78 M
80 M
22 F
PT (sec)
11.5–14.5
13.3
13.2
13.0
12.6
13.0
12.9
12.6
APTT (sec)
29–38
44
48
41
48
46
34.4
33.6
APTTmix1:1
(0`, 120`)
Correction
Correction
Correction
Correction
Correction
NA
NA
VWF:Ag (%)
50–160
25
61
83
102
29
269
35
VWFGPIbM (%)
46–146 (0 group)
61–179 (non-0)
14
34
29
62
11
174
41
VWFGPIbM/Ag ratio
˃ 0.7
0.56
0.55
0.35
0.61
0.38
0.65
1.25
FVIII:C %
60–150
42
37
48
118
21
253
65
RISTOhigh (U)
98–180
12
ND
ND
151
38
ND
112
WBC count 109 /L
4–10
5.6
14.9
12.5
15.2
4.2
8.1
7.7
RBC count 1012 /L
M 4.5–6.0;
N 4.0–5.5
4.6
5.4
8.5
5.7
5.0
6.2
4.1
Hematocrit (%)
M 40–52;
N 36–47
40
46
50
47
46
57
38
Platelet count 109 /L
150–400
245
1391
1120
785
224
142
326
VWF multimers
Persons without VWD (21): Normal distribution
Loss of HMWM
Decrease of HMWM
Loss of HMWM
Decrease of HMWM
Decrease of HMWM
Decrease of HMWM
Normal distribution
LMWM (%)
15.3 (11–23)
50.9
33.1
58.3
32.5
49.1
35.1
13.8
IMWM (%)
30.2 (23.1–35.8)
38.3
39.4
33.5
39.1
19.1
35.9
25.0
HMWM (%)
54.8 (45.1–65.9)
10.8
27.5
8.3
28.4
31.8
29.0
61.2
Case 1. A 67-year-old female patient with a diagnosis of NHL from 2012 onward was
referred for consultation with a suspected bleeding disorder. Three bleeding episodes
were noted during a period of 6 months before a definite AVWS diagnosis was made.
First, a severe bleeding episode had occurred related to puncture of the right maxillary
sinus; then, 3 months later, she was admitted to the emergency department due to recurrent
bleeding after tooth extraction requiring tamponade and bleeding from the right nasal
cavity requiring electrocauterization. The patient was treated with tranexamic acid
during all bleeding events and continues to be followed-up in the hematology clinic.
Case 2. A 33-year-old female patient with heavy menorrhagia and high platelet count
was investigated. She had no antithrombotic treatment. A diagnosis of ET with a positive
finding for a JAK2 (V617F) mutation was made. Menorrhagia was caused by secondary von Willebrand syndrome,
and treatment with tranexamic acid was prescribed for use during menstrual bleeding.
Case 3. A 61-year-old female patient was investigated after experiencing bleeding
after tooth extraction lasting 2 days. A high blood platelet count suggested the possibility
of chronic myeloproliferative disease together with secondary von Willebrand syndrome.
Further investigations confirmed JAK2 (V617F)-positive ET. Cessation of bleeding symptoms was achieved after platelet count
normalization with hydroxyurea treatment.
Case 4. A 60-year-old female patient with PV from 2000 onward was referred for additional
examination and consultation before planned tooth extraction. She experienced bleeding
complications 2 year earlier after the tooth extraction. She was treated with hydroxyurea,
blood exfusion, and low-dose aspirin. She was advised to stop aspirin 5 days before
her next planned tooth extraction. Prophylactic treatment with 10 mg/kg of tranexamic
acid given intravenously (IV) was prescribed three times daily on the procedure day
and also one day before and after the procedure.
Case 5. A 78-year-old male patient was consulted because of recurrent epistaxis, with
a need for cauterization throughout 2 previous years. His complete blood count was
normal. Biochemical investigation showed a monoclonal peak (3.1 g/L) in the γ-globulin
region. Immunoglobulin G kappa monoclonal protein was confirmed by immunofixation.
The kappa/lambda free light-chain ratio was 5.2 (reference range: 0.26–1.65), compatible
with a diagnosis of MGUS. Tranexamic acid was prescribed in the case of a bleeding
episode and the patient remains under close follow-up observation by the hematology
clinic.
Case 6. An 81-year-old male patient with cardiovascular disease and obstructive sleep
apnea syndrome was referred to a hematologist by his general practitioner due to frequent
epistaxis (nosebleeds) occurring in the 2 previous years, with the need for nasal
tamponade at the emergency department. The complete blood count revealed an increased
red blood cell count (6.1012 /L), increased hemoglobin level (176 g/L), and increased hematocrit concentration
(54.9%), which raised the suspicion for PV. However, further studies on BCR/ABL p210 and JAK2 V617F mutations were normal, supporting the diagnosis of secondary erythrocytosis
due to cardiovascular disease, which is one condition that can cause AVWS. The patient
was counseled, and instructions were given for handling future bleeding episodes.
Tranexamic acid was also prescribed to treat further bleeding episodes.
Case 7. A 22-year-old female patient was referred to the hematologist for bleeding
evaluation. She reported the development of apparently spontaneous subcutaneous hematomas,
unrelated to trauma or physical activity, during the last 3 years. Additional examination
showed increased thyroid-stimulating hormone (TSH) and thyroid peroxidase (˃ 1000
U/mL) levels, consistent with a diagnosis of autoimmune thyroiditis, and the patient
was referred to the endocrinologist. Her hypothyroidism was treated and, 1 year later,
normal TSH values were recorded together with normalization of coagulation test findings
for VWF:Ag (69%), VWF:GPIbM (86%), fibrinogen (2.58 g/L), and CRV (< 1 mg/L).
Results
Coagulation Workup for AVWD Diagnosis
In this case series, coagulation studies showed normal PT and prolonged APTT (Cases
1–5). Mixing study revealed corrections for both immediate and incubated APTT tests,
indicating a mild deficiency of FVIII in Cases 1, 2, 3, and 5. FIX, FXI, and FXII
levels were normal. Follow-up assessments demonstrated severely decreased (< 35%)
VWF activity in four of seven patients ([Table 1 ]), fulfilling the criteria for VWD diagnosis. Both decreased VWF:Ag and VWFGPIbM
levels in Cases 1 and 5 and normal VWF:Ag levels with low VWF:GPIbM levels in Cases
2 and 3 were observed. In patient 6, the levels of VWF:Ag, VWFGPIbM, and FVIII:C were
increased, while a decreased VWF function/antigen ratio (VWFGPIbM/VWF:Ag) was recorded.
High-dose ristocetin-induced platelet aggregation was decreased in two patients (cases
1 and 5), while low-dose ristocetin-induced platelet aggregation was normal. In Case
7, the levels of VWF:Ag and VWFGPIbM were both decreased with a normal VWF function/antigen
ratio. Complete blood count and platelet aggregation studies were normal.
VWF multimeric analysis (
[Figs. 1 ] B-F ) revealed decreased HMW multimers, supporting AVWS in all instances (Cases 1–6).
In Case 6, during the visual investigation of gel, we did not detect any abnormalities
in the VWF pattern, yet densitometric data provided additional information about the
VWF multimeric structure. Multimeric analysis (
[Fig. 1 ] H ) showed a normal distribution pattern, suggesting type 1 AVWS. We noted that patients
with lower HMW multimers by densitometric evaluation presented with more severe bleeding
complications.
Fig. 1 Electrophoresis gels and densitograms: A—healthy person, B—Case 1, C—Case 2, D—Case
3, E—Case 4, F—Case 5, G—Case 6, H—Case 7. LMWM, low-molecular-weight multimers; IMWM,
intermediate-molecular-weight multimers; HMWM, high-molecular-weight multimers; PNP,
pool normal plasma.
Discussion
We herein describe the clinical and laboratory data of seven patients with AVWS. All
cases were discussed in a multidisciplinary meeting involving both clinical and laboratory
experts. In all cases, the laboratory findings and lack of previous lifelong bleeding
episodes and family history suggested AVWS.
Earlier studies have documented that MGUS,[13 ]
[14 ]
[15 ] NHL,[16 ] ET,[17 ]
[18 ] and autoimmune hypothyroidism[19 ] are associated with AVWS. The pathogenesis of AVWS is variable but may have an overlapping
mechanism among patients with different underlying disorders.[4 ]
[20 ]
[21 ]
In our series, six patients showed a type 2–like phenotype with decreased VWF activity
to the Ag ratio and a loss/decrease of HMW multimers. One patient had a type 1 VWD
phenotype. Recently developed diagnostic algorithms, based on standard laboratory
assays, may assist clinicians in the diagnostic workup and help differentiate between
AVWS and VWD types 1 and 2. As reported by Federici et al, using data from the AVWS
2004 International Registry,[22 ] AVWS is often correlated with a reduced ratio of VWF:RCo/VWF:Ag. The same findings
were observed in our study in all cases except in Case 7, where the patient’s clinical
presentation was caused by the decreased synthesis of VWF.
Notably, our study demonstrated that VWF multimer analysis aids in the diagnosis of
AVWS as an important, valuable tool. We further observed in our study that decreases
in the level of VWFGPIbM and ratio of VWFGPIbM /VWF:Ag were associated with the selective
loss or decrease of HMW multimers. Our study is in agreement with the report by Tiede
et al,[23 ] which suggested that a reduced VWF:RCo/Ag ratio in AVWS indicates inhibitory antibodies
or a selective loss or decrease in HMW multimers. Separately, research conducted in
Germany[24 ]
[25 ] reported that, in 207 patients with cardiovascular disorders associated with AVWS
and a loss of the HMW multimers, only 44% showed a ratio below 0.7 and noted that
those patients would have likely been misdiagnosed without multimer analysis. In addition,
VWF multimer analysis has been reported by Chen and Nichols as the most sensitive
and specific method available for detecting such AVWS or acquired VWF abnormality
without definite bleeding symptoms.[26 ] In line with a previous study,[27 ] our results revealed that AVWS is also associated with a decreased response to the
higher ristocetin concentration (Cases 1 and 5).
The incidence of AVWS is possibly underestimated in the clinic. For example, as seen
in the retrospective report by Mital et al on ET patients, AVWS may develop as frequently
as in every fifth patient with ET.[28 ] Furthermore, AVWS should be considered in all patients with new-onset bleeding whenever
the laboratory findings suggest VWD, particularly in the presence of an AVWS-associated
disorder. AVWS testing is also recommended prior to surgery or an intervention characterized
by a high risk of bleeding in any individual with an AVWS-associated disorder. Treatment
of the patient’s underlying condition can lead to remission of AVWS. Strategies to
prevent and/or treat bleeding episodes should also be put into place, including the
use of VWF-containing FVIII concentrates, desmopressin, and tranexamic acid. Treatment
success will depend largely upon the underlying pathogenesis of the disorder. Therefore,
investigation of the VWF multimers presents profound clinical significance in suspected
AVWS.
The gold standard for the detection of structural abnormalities of VWF is the multimeric
assay.[29 ] We assessed the VWF multimeric pattern in gels and quantified multimeric fractions
using Sebia analysis software program. This method is easy to use and could prove
very useful in future laboratory workup required for the diagnosis of AVWS. Interestingly,
the densitometric evaluation of VWF multimers showed that patients with lower HMW
multimer values presented with more severe bleeding complications. However, most AVWS
patients do not bleed until they experience additional triggers like invasive procedures
or trauma.[4 ] Therefore, correct identification of patients with AVWS is a prerequisite for determining
the applicable guidance on clinical management.[30 ]
