Expanding your Choices: Recent additions to the VWF test menu

Transcription

1 Expanding your Choices: Recent additions to the VWF test menu Kenneth Friedman, M.D. Director, Hemostasis Reference Lab BloodCenter of Wisconsin, Milwaukee WI

2 Disclosures for Ken Friedman, M.D. Research Support/P.I. Employee Consultant Major Stockholder Speakers Bureau Honoraria Scientific Advisory Board No relevant conflicts of interest to declare BloodCenter of Wisconsin Ablynx, Bayer, CSL Behring, Genentech, Novo Nordisk, Shire No relevant conflicts of interest to declare Alexion No relevant conflicts of interest to declare No relevant conflicts of interest to declare

3 Agenda 1. Two brief cases 2. Emerging assays GPIbM for VWF-platelet binding activity VWF collagen binding activity VWF propeptide antigen to assess VWF clearance VWF sequence analysis 3. Case comments and Summary

4 Polling 12 yo Question with hematoma after playing volleyball Patient: 12 yo African American female developed a large wrist hematoma shortly after playing volleyball at school. She has had no bleeding issues in the past, and denies menorrhagia. Family history indicates mother has easy bruising and was diagnosed as having type 2M von Willebrand disease. Reference Interval VWF:Ag 60 IU/dL [ IU/dL] VWF:RCo 35 IU/dL [ IU/dL] FVIII 90 IU/dL [ IU/dL] VWF multimer Normal Platelet Count 311,000 [150, ,000] Blood Group O The next appropriate test to perform is? A. VWF collagen binding assay B. Ristocetin-induced platelet aggregation C. VWF:GPIbM assay D. VWF full gene sequence analysis 4

5 Polling 18 yo Question needs dental extraction Patient: 18 yo female with history of menorrhagia, easy bruising, and iron deficiency anemia; menorrhagia is controlled with OCP. Patient was referred to oral surgeon for wisdom teeth extraction. Given the history, oral surgeon referred this patient to hematology, where the following labs were obtained: Reference Interval VWF:Ag 14 IU/dL [ IU/dL] VWF:RCo 16 IU/dL [ IU/dL] FVIII 16 IU/dL [ IU/dL] VWF:CB 15 IU/dL [ IU/dL] Platelet Count 311,000 [150, ,000] Blood Group O What additional diagnostic testing is indicated before DDAVP trial? A. VWF multimer B. VWF propeptide C. Low-dose ristocetin platelet aggregation D. None, do the trial 5

6 What Is VWF? Multimeric glycoprotein synthesized in endothelial cells and megakaryocytes (platelets) Sadler, Ann Rev Biochem 1998;67:395.

7 VWF: an adapter protein Platelets VWF Clotting factors Factor VIII Vessel wall Collagen

8 Von Willebrand Factor (VWF) VWF has 2 main functions 1. Chaperone factor VIII in plasma 2. Platelet adhesion at sites of vascular injury VWF binds to subendothelial collagen VWF captures platelets via glycoprotein Ib N propeptide (VWFpp) S D1 D2 D D3 A1 A2 A3 D4 B1 B2B3 C1 C2 S C Directs intracellular processing FVIII GPIb collagen collagen ADAMTS13 cleavage site RGDS GPIIb/IIIa

9 Classification of Von Willebrand Disease Inherited VWD Type 1 Partial quantitative VWF deficiency Prevalence in VWD ~85% Type 2 Qualitative VWF defect Four subtypes: 2A, 2B, 2M, 2N Prevalence in VWD ~ 15-20% Type 3 Virtually complete VWF deficiency and decreased factor VIII Prevalence ~ 1 in 10 6 individuals Acquired VWD Ng, C, Motto DG, Di Paola J. Blood 2015;125:

10 Initial Patient Evaluation in VWD VWD Guideline NHLBI Expert Panel No single assay is sufficient Quantitative assays VWF antigen Is VWF present? VWF ristocetin cofactor activity Does VWF bind platelets? Factor VIII activity Is Factor VIII present in normal amount? Functional Ratios VWF:RCo/VWF:Ag & FVIII/VWF:Ag Nichols WL, et al. Haemophilia. 2008;14:

11 Prototype Laboratory Data VWD Guideline NHLBI Expert Panel Condition VWF:RCo VWF:Ag FVIII RCo/Ag FVIII/Ag Low or 1* <50* <50* or NL Normal Normal 2A or 2M <30* <30 200* or NL Normal 2B <30* <30 200* or NL Normal 2N Normal Low 3 <3 <3 < * <30 was suggested to classify definitive VWD - Term Low VWF Classification: quantitative deficiency with VWF level IU/dL Nichols WL, et al. Haemophilia. 2008;14:

12 Supplemental Assays Additional testing depends on initial results Multimer distribution Are large multimers missing? VWF collagen binding (VWF:CB) Another VWF function and surrogate for multimer assay VWF propeptide (VWFpp) Is VWF synthesis and survival normal? VWFpp/VWF:Ag ratio Specific binding assays Is ristocetin driven VWF-platelet enhanced (LD-RIPA)? Is Factor VIII binding function normal? VWF gene sequencing Ng, C, Motto DG, Di Paola J. Blood 2015;125:

13 Prototype Laboratory Data VWD Guideline NHLBI Expert Panel Condition VWF:RCo VWF:Ag FVIII RCo/Ag FVIII/Ag Low or 1* <50* <50* or NL Normal Normal 2A or 2M <30* < * or NL Normal 2B <30* < * or NL Normal 2N Normal Low 3 <3 <3 < VWF:RCo/Ag ratio is problematic Nichols WL, et al. Haemophilia. 2008;14:

14 Limitations of Screening Panel VWF:RCo is a problematic assay 1. Ristocetin cofactor (VWF:RCo) 1 : Lower limit of detection: Wide range 3-20 IU/dL Considerable assay imprecision 2. Ratio of VWF:RCo/VWF:Ag Poor performance of VWF:RCo assay limits the utility of ratio As the VWF:Ag falls, the discriminative value worsens 3. Ristocetin activation of VWF is flawed 2 Polymorphism p.d1472h shows low VWF:RCo activity Underestimates VWF-platelet binding activity May lead to erroneous diagnosis of VWD 1 Multiple authors, Sem Thromb Hemost 2006; 32: Flood VH, et al. Blood 2010; 116:

15 Interaction of VWF With Platelets Polymorphisms interfere with ristocetin binding A B How common is D1472H? - African Americans: 63% D1472H+ - Caucasians: 17% D1472H+ Flood VH, et al. Blood 2010; 116: Reprinted with permission from Flood VH, et al. Blood. 2010;116:

16 VWF GPIbM Activity Innovance VWF Assay Normal GPIb Mutant GPIb Gain-of Function variants Patzke J, et al. Blood Coagul Fibrinolysis. 2014;25:

17 VWF GPIbM Activity Method comparison between Innovance vwf:gpibm and vwf:rco vwf:gpibm vwf:rco Patzke J, et al. Blood Coagul Fibrinolysis. 2014;25:

18 VWF GPIbM Activity Method comparison of von Willebrand disease samples between Innovance vwf:gpibm and vwf:rco vwf:gpibm vwf:rco Reproduced with permission from Patzke J, et al. Blood Coagul Fibrinolysis. 2014;25:

19 VWF:GPIbM Activity Assay Principle OPD, 30% Hydrogen Peroxide Y VWF 2 Antibody-HRP 1 Antibody: Polyclonal anti-vwf Diluted Patient Sample GP1bM Y Recombinant GPIbM Anti-VWF MoAb 19

20 Interaction of VWF With Platelets Polymorphisms interfere with ristocetin binding Flood VH, et al. Blood. 2010;116:

21 VWF:GPIbM Activity Assay Summary VWF:GPIbM activity assay provides a measure of VWF platelet binding activity independent of ristocetin VWF:GPIbM is not affected by the D1472H VWF polymorphism that may result in a false low VWF:RCo activity and a potential misdiagnosis of type 2M CVs for VWF:GPIbM activity assay are low Innovance 2% 7% BloodCenter plate method 15% Lower limit of detection for VWF:GPIbM activity assay is 2.0 u/dl Still not a physiologic assay - Does not use shear to induce VWF-platelet interaction 21

22 Prototype Laboratory Data VWD Guideline NHLBI Expert Panel Condition VWF:RCo VWF:Ag FVIII RCo/Ag FVIII/Ag Low or 1* <50* <50* or NL Normal Normal 2A (Short) <30* < * or NL Normal 2M (Point Mutation) <30* < * or NL Normal 2B (Short) <30* < * or NL Normal 2N Normal Low 3 <3 <3 < VWF Multimer assay is problematic Assay is cumbersome with slow turn-around times Modified from Nichols WL, et al. Haemophilia. 2008;14:

23 VWF Multimer Distribution Are the large multimers present? Is the spectrum of multimers is normal? Abnormal in Types 2A, 2B, platelet-type and acquired VWD Absent in type 3 VWD But: Assay is cumbersome with slow turn-around-time Flood VH et al, Clin Chem 2013 Apr;59(4):684-91

24 VWF Collagen-Binding Activity Another assay of VWF function Quantitative assay based upon preferential binding of larger VWF multimers to collagen - VWF A3 domain binds collagen types I and III - (VWF A1 domain binds collagen types IV and VI) Assay characteristics ELISA format Low limit of detection and CV is similar to VWF:Ag VWF * Color ELISA, enzyme-linked immunosorbent assay 24

25 VWF Collagen-Binding Activity A sensitive screen of multimer distribution The ratio of VWF:CB/VWF antigen is a highly sensitive screen for a defect of VWF multimer distribution Flood VH, et al. Clin Chem. 2013;59: ; 2. Adcock D. Semin Thromb Hemost. 2006;32: ; 3. Favaloro EJ. Semin Thromb Hemost. 2009;35:

26 VWF Collagen-Binding Assay Summary VWF:CB/VWF:Ag ratio is highly sensitive to multimer structure and may be used to predict multimer defects 1-3 Collagen binding assays also evaluate a discrete VWF function Specific defects of collagen binding in patients with normal multimer distribution have been identified: Type 2M-C 4-6 Collagen binding defects may contribute to bleeding risk We don t find these defects if we don t look for them 1. Flood VH, et al. Clin Chem. 2013;59: ; 2. Adcock D. Semin Thromb Hemost. 2006;32: ; 3. Favaloro EJ. Semin Thromb Hemost. 2009;35:60-75; 4. Pareti FI, et al. J Biol Chem. 1987;262: ; 5. Rand JH, et al. J Clin Invest. 1991;88: ; 6. Flood VH, et al. J Thromb Haemost. 2012;10:

27 Prototype Laboratory Data VWD Guideline NHLBI Expert Panel Condition VWF:RCo VWF:Ag FVIII RCo/Ag FVIII/Ag Low or 1* Low Production Type 1C Fast clearance <50* <50* or NL Normal Normal <30 <30 Normal Normal 2A <30* <30 200* or NL Normal 2M <30* < * or NL Normal 2B <30* < * or NL Normal 2N Normal Low 3 <3 <3 < Distinguishing Type 1 from Type 1C is important for treatment Modified from Nichols WL, et al. Haemophilia. 2008;14:

28 Response to DDAVP: VWD Type 1 Two patients with different response profiles VWF:Ag (IU/dL) Time (hours) Decreased Synthesis Rapid Clearance Gill JC. Personal communication 28

29 Type 1C VWD Due to increased VWF clearance Mechanism Short half-life of VWF Laboratory findings are subtly different from type 1 VWD Factor VIII is reduced to a similar extent as VWF:Ag Initial hyper-fold rise of VWF in response to desmopressin But VWF elevation not sustained, half-life only 1 4 hours (VWF replacement provides more sustained VWF levels) Haberichter SL, et al. Blood. 2008;111:

30 VWFpp/VWF:Ag Patients With Type 1C Mutations Increased VWFpp/VWF:Ag identifies patients with increased VWF clearance Haberichter SL, et al. Blood. 2006;108:

31 Type 1VWD The Majority of Severe Type 1 VWD Cases are 1C Percentage consistent with type 1C phenotype % 38% 7% Increased Clearance VWFpp/VWF:Ag Reduced Secretion VWF:Ag (IU/dL) Haberichter SL. Personal communication 31

32 VWFpp Assay Summary VWFpp/VWF:Ag ratio is a marker of VWF clearance VWFpp is a useful marker to identify type 1C and acquired VWD Type 1C VWD compromises a substantial proportion of the cases of Type 1 VWD when the VWF antigen is <20 IU/dL Treatment is different for patients with type 1C Replacement with VWF concentrate provides more sustained factor levels than DDAVP treatment 32

33 Prototype Laboratory Data VWD Guideline NHLBI Expert Panel Condition VWF:RCo VWF:Ag FVIII RCo/Ag FVIII/Ag Low or 1* <50* <50* or NL Normal Normal 2A (Short) <30* < * or NL Normal 2M (Point Mutation) <30* < * or NL Normal 2B (Short) <30* < * or NL Normal 2N Normal Low 3 <3 <3 < Type 2N VWD requires differentiation from hemophilia A Modified from Nichols WL, et al. Haemophilia. 2008;14:

34 2N VWD: DDAVP Trial Poor Shortened F-VIII response Courtesy: Ralph Gruppo, MD

35 Factor VIII Binding Assay VWF:FVIIIB 1.2 S2222 Color rfviii color * FVIII/VWF bound type 2N/2N type 2N/1 type 2N/nl normal

36 VWF Domain Structure and Locations of Mutations SP Propeptide Mature VWF Furin N S-S Exon 28 B D1 D2 D' D3 A1 A2 A3 D4 C1 C2 CK S-S C FVIII GPIb Collagen RGDS GPIIb/IIIa ADAMTS13 cleavage N B D1 D2 D' D3 A1 A2 A3 D4 C1 C2 Type 2A * * ** **** * Type 2B **** Type 2M **** * Type 2N ** **** Type 1/3 ** * ** *** ** * *** ** ** *** * * ** ** *** * * * * * * * * Type 1c CK C

37 In the age of genetic diagnosis Good phenotype data is still very important Phenotype data is still key Lab phenotype explains symptoms and drives test algorithms Genetic testing has a role: Usually identifies sequence variations responsible for type 2 VWD Confirm diagnosis for type 2M VWD variants Distinguish overlapping phenotypes 2A vs 2B 2B vs Platelet-type 2N vs Hemophilia A 1C vs 3 Determines risk for inhibitor and recurrence risk in type 3 families Limitations of genetic analysis Multiple mechanisms underlie quantitative VWF deficiencies Yield is lower when VWF level > 30 IU/dL 37

38 Polling 12 yo Question with hematoma after playing volleyball Patient: 12 yo African American female developed a large wrist hematoma shortly after playing volleyball at school. She has had no bleeding issues in the past, and denies menorrhagia. Family history indicates mother has easy bruising and diagnosed in as having type 2M von Willebrand disease. Reference Interval VWF:Ag 60 IU/dL [ IU/dL] VWF:RCo 35 IU/dL [ IU/dL] FVIII 90 IU/dL [ IU/dL] VWF multimer Normal Platelet Count 311,000 [150, ,000] Blood Group O The next appropriate test to perform is? A. VWF collagen binding assay B. Ristocetin-induced platelet aggregation C. VWF:GPIbM assasy D. VWF full gene sequence analysis (just exon28) 38

39 Polling 18 yo Question needs dental extraction Patient: 18 yo female with history of menorrhagia, easy bruising, and iron deficiency anemia; menorrhagia is controlled with OCP. Patient was referred to oral surgeon for wisdom teeth extraction. Given the history, oral surgeon referred this patient to hematology, where the following labs were obtained: Reference Interval VWF:Ag 14 IU/dL [ IU/dL] VWF:RCo 16 IU/dL [ IU/dL] FVIII 16 IU/dL [ IU/dL] VWF:CB 15 IU/dL [ IU/dL] VWFpp 73 IU/dL [ IU/dL] Platelet Count 311,000 [150, ,000] Blood Group O DDAVP Trial: Pre 1 hr 4 hr VWF:Ag VWF:Rco < FVIII What additional diagnostic testing is indicated before DDAVP trial? A. VWF multimer B. VWF propeptide C. Low-dose ristocetin platelet aggregation D. None, do the trial - The high VWFpp/VWF:Ag ratio suggests VWD type 1C - The DDAVP trial should include lab draws at both 1 hour and 4 hours post-ddavp to assess the magnitude and duration of response after DDAVP 39

40 Conclusions - 1 Classification of VWD remains complex; utilization of new VWF assays may aid in proper VWD diagnosis, providing direction for optimal treatment strategies VWF:GPIbM assay is an improvement over VWF ristocetin cofactor assay, with better sensitivity for low VWF levels, tighter CV, and lack of interference due to polymorphism p.d1472h Collagen binding assay evaluates a discrete function of VWF and is an excellent screen for VWF multimer defects Assay of VWFpp can identify type 1C VWD, a common condition among type 1 patients with VWF:Ag <20 40

41 Conclusions - 2 VWD Type 2N can be distinguished from mild hemophilia A via the VWD Type 2N binding assay VWF sequence analysis is most useful to distinguish type 2 variants in situations where phenotype assays are insufficient. 41