THE DIAGNOSIS

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THE DIAGNOSIS OF VON WILLEBRANDS
DISEASE Mareli Kelderman, Muriel Meiring, Philip
Badenhorst Haematology and Cell Biology,
University of the Free State NHLS,
Bloemfontein

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VON WILLEBRAND DISEASE
? bleeding disorder caused by a deficiency or
an abnormality of VWF
? VWD patients may have mild, moderate to
severe bleeding, usually proportionate to degree
of VWF defect
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VON WILLEBRAND FACTOR

• plasma proteins that play an important role in
  haemostasis and blood coagulation
• promote platelet adhesion to sub-endothelium
• form thrombi
• carry FVIII
• protect FVIII

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correct diagnosis of types and subtypes for
appropriate therapy
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CLASSIFICATION OF TYPES AND SUBTYPES
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• LABORATORY DIAGNOSIS
• Clinical History
• 2. Prescribed tests
• ? Von Willebrand Antigen
• (VWFAg)
• ? Ristocetin Cofactor
• (Ricof)
• ? Factor VIII
• ? Blood Grouping
• ? Multimer Analysis
• ? RIPA (Ristocetin
• induced platelet
• agglutination)
• ? Factor VIII Binding
• Assay
• ? Bleeding Time
• ? Full Blood Count

REFERENCE
Federici, A.B., Castaman, G., Mannucci, P.M.
2002. Guidelines for the diagnosis and
management of von Willebrand disease in Italy.
Haemophilia, vol. 8, pp. 607-621.
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LABORATORY DIAGNOSIS 1. Clinical History 2.
Prescribed tests ? Von Willebrand
Antigen (VWFAg) ? Ristocetin Cofactor
(Ricof) ? Factor VIII ? Blood
Grouping ? Multimer Analysis ?
RIPA (Ristocetin induced platelet
agglutination) ? Factor VIII Binding
Assay ? Bleeding Time ? Full
Blood Count (platelet count) ?
Coagulation Tests (PT, PTT)
MULTIMERS
RIPA
FVIII BINDING TEST
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Multimer Analysis
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A Rapid Method to Visualize von Willebrand Factor
Multimers by Using Agarose Gel
REFERENCE Krizek, D.R., Rick, M.E. 2000. A
Rapid Method to Visualize von Willebrand Factor
Multimers by Using Agarose Gel Electrophoresis,
Immunolocalization and Luminographic Detection.
Thrombosis Research, vol. 97, pp. 457-462.
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RIPA (Ristocetin induced agglutination)
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RIPA RESULTS EXPRESSED AS
The Ristocetin concentration that gives an
agglutination of 30
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Factor VIII Binding Assay
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Factor VIII Binding Assay
RESULTS EXPRESSED AS A percentage compared to
that of normal plasma. WithType 2N VWFD the value
is expected to be very low.
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Plasma VWFAg 87
Absent
Type 3
LABORATORY DIAGNOSIS 1. Clinical History
2. Prescribed tests ? Von
Willebrand Antigen (VWFAg) 87 ?
Ristocetin Cofactor (Ricof) 124 ?
Ratio of (Ricof)124 (VWFAg)55 0.8
? Factor VIII 130 ? Blood Grouping
? Multimer Analysis HMW Multimers present
? RIPA (Ristocetin induced platelet
agglutination) 30 agglutination at
1mg/ml Ristocetin ? Factor VIII Binding
Assay 115 ? Bleeding Time ?
Full Blood Count (platelet count) ?
Coagulation Tests (PT, PTT)
Present
Type 1
Proportionate (0.7-1.2)
Proportionate
Plasma FVIII vs VWFAg
Plasma VWFRco vs VWFAg 1.4
124 87
Discrepant
Type 2
Discrepant (lt0.7)
Type 2N
Ristocetin induced Platelet Agglutination R.I.P.A.
(mg/ml)
Increased (0.2-0.8)
Type 2A
Absent
Decreased (gt1.2))
plasma High Multimers
Type 2M
Type 2B
Present
BINDING 115
Multimers present
15
Plasma VWFAg 55
Absent
Type 3
LABORATORY DIAGNOSIS 1. Clinical History
2. Prescribed tests ? Von Willebrand
Antigen (VWFAg) 55 ? Ristocetin
Cofactor (Ricof) 44 ? Ratio of
(Ricof)44 (VWFAg)55 0.8 ?
Factor VIII ? Blood Grouping ?
Multimer Analysis HMW Multimers present
? RIPA (Ristocetin induced platelet
agglutination) ? Factor VIII Binding
Assay ? Bleeding Time ? Full
Blood Count (platelet count) ?
Coagulation Tests (PT, PTT)
Present
Type 1
Proportionate (0.7-1.2)
Proportionate
Plasma FVIII vs VWFAg
Plasma VWFRco vs VWFAg 0.8
44 55
Discrepant
Type 2
Discrepant (lt0.7)
Type 2N
Ristocetin induced Platelet Agglutination R.I.P.A.
(mg/ml)
Increased (0.2-0.8)
Type 2A
Absent
Decreased (gt1.2))
plasma High Multimers
Type 2M
Type 2B
Present
COMMENTS VWF multimer analysis shows a decrease
in multimer numbers but retaining a normal
pattern. This picture is compatible with Type 1
VWD.
Multimers present
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Plasma VWFAg 55
Absent
Type 3
LABORATORY DIAGNOSIS 1. Clinical History
2. Prescribed tests ? Von Willebrand
Antigen (VWFAg) 55 ? Ristocetin
Cofactor (Ricof) 44 ? Ratio of
(Ricof)44 (VWFAg)55 0.8 ?
Factor VIII 20 (ratio 0.4) ? Blood
Grouping ? Multimer Analysis HMW
Multimers present ? RIPA (Ristocetin
induced platelet agglutination)
? Factor VIII Binding Assay 10 ?
Bleeding Time ? Full Blood Count
(platelet count) ? Coagulation Tests (PT,
PTT)
Present
Type 1
Proportionate (0.7-1.2)
Proportionate
Plasma FVIII vs VWFAg 0.4
Plasma VWFRco vs VWFAg 0.8
44 55
Discrepant
Type 2
Discrepant (lt0.7)
Type 2N
Ristocetin induced Platelet Agglutination R.I.P.A.
(mg/ml)
Increased (0.2-0.8)
Type 2A
Absent
Decreased (gt1.2))
plasma High Multimers
Type 2M
Type 2B
Present
COMMENTS VWF multimer analysis shows a decrease
in multimer numbers but retaining a normal
pattern. The ratio of the FVIII vs the VWF is
discrepant. The FactorVIII Binding Assay is
decreased. This picture is compatible with Type
2N VWD.
Multimers present
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Absent
Type 3
Plasma VWFAg 41
LABORATORY DIAGNOSIS 1. Clinical
History 2. Prescribed tests ? Von
Willebrand Antigen (VWFAg) 41 ?
Ristocetin Cofactor (Ricof) 12 ? Ratio
of (Ricof)12 (VWFAg)41 0.29 ?
Factor VIII 55 ? Blood Grouping O
Positive ? Multimer Analysis HMW
Multimers absent ? RIPA (Ristocetin
induced platelet agglutination)
? Factor VIII Binding Assay ? Bleeding
Time ? Full Blood Count (platelet
count) ? Coagulation Tests (PT, PTT)
Present
Proportionate
Type 1
Proportionate (0.7-1.2)
Plasma FVIII vs VWFAg
Plasma VWFRco vs VWFAg 0.29
12 41
Discrepant
Type 2
Discrepant (lt0.7)
Type 2N
Increased (0.2-0.8)
Ristocetin induced Platelet Agglutination R.I.P.A.
(mg/ml)
Type 2A
Absent
Decreased (gt1.2)
plasma High Multimers
Type 2M
Type 2B
Present
COMMENTS Multimeric analysis shows an absence
of high molecular weight multimers as seen in 2A
Von Willebrands Disease. A RIPA test is
indicated to exclude Type 2B disease.
HMW Multimers absent
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Absent
Type 3
Plasma VWFAg 41
LABORATORY DIAGNOSIS 1. Clinical
History 2. Prescribed tests ? Von
Willebrand Antigen (VWFAg) 41 ?
Ristocetin Cofactor (Ricof) 12 ? Ratio
of (Ricof)12 (VWFAg)41 0.29 ?
Factor VIII 55 ? Blood Grouping O
Positive ? Multimer Analysis HMW
Multimers absent ? RIPA (Ristocetin
induced platelet agglutination)
Ristocetin 0.4 mg/ml ? ? Factor VIII
Binding Assay ? Bleeding Time ?
Full Blood Count (platelet count) ?
Coagulation Tests (PT, PTT)
Present
Proportionate
Type 1
Proportionate (0.7-1.2)
Plasma FVIII vs VWFAg
Plasma VWFRco vs VWFAg 0.29
12 41
Discrepant
Type 2
Discrepant (lt0.7)
Type 2N
Increased (0.2-0.8)
Ristocetin induced Platelet Agglutination R.I.P.A.
(mg/ml) 0.6
Type 2A
Absent
Decreased (gt1.2)
plasma High Multimers
Type 2M
Type 2B
Present
COMMENTS Multimeric analysis shows an absence
of high molecular weight multimers. The increased
RIPA test indicates Type 2B Von Willebrands
Disease.
HMW Multimers absent
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Plasma VWFAg 77
Absent
Type 3
LABORATORY DIAGNOSIS 1. Clinical
History 2. Prescribed tests ?
Von Willebrand Antigen (VWFAg) 77 ?
Ristocetin Cofactor (Ricof) 30 ? Ratio
of (Ricof)30 (VWFAg)77 0.39 ?
Factor VIII 99 ? Blood Grouping
? Multimer Analysis HMW Multimers present
? RIPA (Ristocetin induced platelet
agglutination) Ristocetin 1.25 mg/ml ? ?
Factor VIII Binding Assay ? Bleeding Time
? Full Blood Count (platelet count)
? Coagulation Tests (PT, PTT)
Present
Type 1
Proportionate (0.7-1.2)
Proportionate
Plasma FVIII vs VWFAg
Plasma VWFRco vs VWFAg 0.4
30 77
Discrepant
Type 2
Discrepant (lt0.7)
Type 2N
Ristocetin induced Platelet Agglutination R.I.P.A.
?
Increased (0.2-0.8)
Type 2A
Absent
Decreased (gt1.2)
plasma High Multimers
Type 2M
Type 2B
Present
COMMENTS Discrepant RC/Ag ratio indicative of a
Type 2 Von Willebrands Disease. A decreased RIPA
and the presence of multimers suggests a Type 2M
disease.
Multimers present
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Plasma VWFAg 102
Absent
Type 3
LABORATORY DIAGNOSIS 1. Clinical
History 2. Prescribed tests ?
Von Willebrand Antigen (VWFAg) 102 ?
Ristocetin Cofactor (Ricof) 26 ? Ratio
of (Ricof)26 (VWFAg)102 0.25 ?
Factor VIII 71 ? Blood Grouping
? Multimer Analysis HMW Multimers present
? RIPA (Ristocetin induced platelet
agglutination) Ristocetin gt1.2 mg/ml ?
Factor VIII Binding Assay ? Bleeding Time
? Full Blood Count (platelet count)
? Coagulation Tests (PT, PTT)
Present
Type 1
Proportionate (0.7-1.2)
Proportionate
Plasma FVIII vs VWFAg
Plasma VWFRco vs VWFAg 0.25
26 102
Discrepant
Type 2
Discrepant (lt0.7)
Type 2N
Ristocetin induced Platelet Agglutination R.I.P.A.
? gt1.2
Increased (0.2-0.8)
Type 2A
Absent
Decreased (gt1.2)
plasma High Multimers
Type 2M
Present
Type 2B
COMMENTS Discrepant RC/Ag ratio indicative of a
Type 2 Von Willebrands Disease. A decreased RIPA
and the presence of multimers suggest a Type 2M
disease.
No concentration gave 30 agglutination
Multimers present
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CLASSIFICATION OF TYPES AND SUBTYPES
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CONCLUSION

• This proves to be a practical and successful
  approach to diagnose VWD
• With the necessary information and a small amount
  of plasma we can assist any laboratory in South
  Africa with the diagnosis and typing of their
  patients

Haematology and Cell Biology, University of the
Free State NHLS, Bloemfontein

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