Upload
others
View
3
Download
0
Embed Size (px)
Citation preview
Results of vWF Questionnaire andResults of vWF Questionnaire andvWF Surveys vWF Surveys -- 20042004
Ellinor I. Peerschke, PhDDonna Castellone, MS, MT(ASCP)SH
On behalf of the PT/EQA Working Group
2004 NASCOLA2004 NASCOLAAnnual Scientific MeetingAnnual Scientific Meeting
2
Members of the PT/EQA Working Group
• Dr Ellinor Peerschke (Chair) Weill/Cornell Medical Center• Dr Larry Brace University of Illinois, Chicago• Ms Donna Castellone New York Presbyterian Hospital• Mr David Chance St Louis Coagulation Consultants• Mr Stephen Johnson Tufts, New England Medical Center• Dr Richard Marlar VA Medical Center, Oklahoma City• Dr John Olson University of Texas Health System• Dr Elizabeth Van Cott Massachusetts General Hospital• Dr James Zehnder Stanford University• Dr William Nichols (ex officio) Mayo Clinic• Ms Marlies Ledford-Kraemer (ex officio) CLOT-ED, Inc
3
Questionnaire Distribution
• 39 surveys distributed by e-mailed• 34 completed surveys returned• Response rate: ~87%
4
Goals /Aims
• Understand laboratory practices
• Understand test strategies
• Understand result interpretation
5
Preanalytical VariablesSample Processing
• Analysis is done on fresh and/or frozen samples
• Blood processed within 0-4 hours (75%)
• Assayed fresh (6%), frozen (24%), either (67%)
• Samples frozen in freezer (91%)
• Thawed at 37oC (94%)• Placed at room
temperature (70%) or ice (27%)
• Assayed between 0-20 (48%), 0-40 (40%) and 40-60 (15%) min after thaw
• Samples tested in batch (91%)
Numbers in parentheses represent percent respondents
6
vWF Testing Panel
vWF Antigen 100%vWF Act 94%F VIII 85%vWF Multimers 35%PFA-100 30%APTT 21%RIPA 15%
CBA: 9%Bleeding Time 6%ABO Typing 6%Fibrinogen 6%CBC 3%APTT Mixing Study 3%Interpretation 9%
Numbers represent percent respondents who perform assay
7
vWF Antigen Method
• Automated: 86%– Latex Agglutination
• ELISA: 11%• Immunoelectrophoresis: 3%
Numbers represent percent respondents
8
vWF Antigen Calibrator
• Stago LIA 53%• ELISA Kit 13%• Precision Biologic Gold
Standard 9%
• Precision Biologic + ISTH 6%
• Cryocheck 6% • In-house PNP 3%• Stago Unical 3%• George King 3%• IL CAL Plasma 3%
Numbers represent percent respondents
9
vWF Antigen CalibratorAssay Value Assignment
• Manufacturer assigned value: 79%• Assayed against WHO Reference: 20%• Assayed relative to previous standard: 6%
Note:23% of respondents assay a WHO standard or reference plasma different from calibrator with each assay
Numbers represent percent respondents
10
vWF Activity Testing
• Ristocetin cofactor activity– Aggregometry: 51%– Automated: 42%– ELISA: 6%– One respondent does not perform vWF activity
Numbers represent percent respondents
11
vWF Activity Calibrator
• Dade SHPL 30%• Precision Biologic
Gold Ref 24%• Unknown 12%• Precision Biologic
Cryocheck 9%• In-house PNP 6%
• Bio Data NRP 3%• Verify RP 3%• Helena SARP 3%• ADI Std 3%• Kit Std 3%• Chronolog RP 3%• George King Fact 3%
Numbers represent percent respondents
12
vWF Activity Assay Buffers
• TBS 33%• Saline 27%• IBS 18%• Kit 6%• HEPES 3%
• PBS 3%• OVB 3%• Modified Tangen’s
buffer with EDTA & sodium azide 3%
• Unknown 3%
Numbers represent percent respondents
13
Ristocetin Concentration
• Reported concentration range:– 1.11ng/ml – 10 mg/ml
• 1.0 mg/ml: 18%• 1.5 mg/ml: 13%
Numbers represent percent respondents
14
vWF Activity Standard Curve
• 65% of laboratories prepare a new curve with each run– All laboratories doing so perform aggregation
• 32% of laboratories prepare a new curve when controls are out of range– Most laboratories doing so use an automated
agglutination method
15
FVIII Activity Calibrator
• Stago Unical 27%• Dade SHPL 21%• Precision Biologic Gold
Reference Pl 12%• BioMerieux VRP 15%
• Precision Biologic CryoCheck 6%
• In-house PNP 6%• IL Cal Plasma 6%• VCRP 3%• Pacific Hemostasis RP
3%• George King Fact 3%
Numbers represent percent respondents
16
FVIII Assay Buffer
• Owrens: 55%• Saline: 18%• IBS: 20%• IL Diluent: 6%• HEPES: 3%
Numbers represent percent respondents
17
FVIII Calibrator CurveNumber of Points
• 6 points: 36% of respondents• 8 points: 23% of respondents• 5 points: 18% of respondents• 7 points: 15% of respondents• 4 points: 9% of respondents
18
FVIII ActivityNumber of Dilutions
Control
• 1 dilution 64%• 2 dilutions 24%• 3 dilutions 6%• 4 dilutions 6%
Patient Plasma
• 3 dilutions 62%• 2 dilutions 21%• 4 dilutions 18%
Numbers represent percent respondents
19
FVIII ActivityInhibitor Effect Definition
• Delta 10% between dilutions (15%)
• Delta 20% between dilutions (15%)
• Delta 15% between dilutions (9%)
• >50% (3%)• Delta 15 U between
dilutions (3%)
• Increasing Concentration (18%)
• Unknown (18%)• Nonparallelism (12%)• Interpretation (6%)• Non Match (3%)• Dilutional (3%)
Numbers in parentheses represent percent respondents
20
ABO Typing Performed as Part of VWF Panel
• Reverse typing: 28%• Forward typing: 13%• Interpretation of vWF studies relative to ABO
group: 41% • Do physicians order ABO typing as part of
vWF studies: 6% Yes, 32% Sometimes
Numbers represent percent respondents
21
Collagen Binding Assay
• 19% of respondents perform collagen binding assay
• Performed in addition to Ristocetin Cofactor assay (some for research purposes only)
22
vWF Multimer Analysis
• 52% of respondents send out samples for multimer analysis
• 35% of respondents perform vWF multimeranalysis on site
23
Criteria for vWFMultimer Testing
All Laboratories• Discordant vWF Antigen to
Activity or Type III: 53%– vWF:Ag / vWF:RCo Ratio
•>2.0: 15%•>1.5: 13%•Not specified: 27%
• Other: New Patient, Low Ag and Act, MD request, contract, Interpretation
Laboratories Performing Multimer Analysis (12)
• None (5)• On request (4)• Abnormal vWF:Ag or
VWF:RCo (2)• Diagnosis of von
WIllebrand Disease (1)
Numbers represent percent respondents Number of respondents
24
vWF Work-up and Multimer Testing
• Percentage of samples submitted for multimer analysis on patients receiving vWF work-ups – 40% of respondents submit <1 - 10% of vWF
work-ups for multimer testing– 30% of respondents submit 10 - 20% of vWF
work-ups for multimer testing– 20% of respondents submit 20 - 41% of vWF
work-ups for multimer testing
25
vWF Multimer AnalysisAgarose
2.011.0
1.510.75
1.3, 1.5, 1.7, 2.070.8
0.60, 0.65, 1.03None
Separating Gel% Agarose Concentration
Number of Labs
Stacking Gel% Agarose Concentration
SDS Agarose Gel Electrophoresis
26
VWF Multimer AnalysisVisualization Methods
• Western Blot: 6 laboratories• Luminescence: 3 laboratories• Autoradiography: 2 laboratories• Unspecified: 1 laboratory
27
VWF Multimer AnalysisVisualization by Western Blot
• Primary antibody source– Polyclonal rabbit anti-human vWF (5)
• DAKO, Diagnostica Stago– Monoclonal mouse anti-human vWF (1)
• Secondary antibody source– Goat anti-rabbit IgG (2-unknown vendor, 1-Promega)– Donkey anti-rabbit IgG (1)– Unknown animal source - BioRad (1)– Goat anti-mouse IgG (1)
• Secondary antibody conjugated with: – Horseradish peroxidase (3)– Biotinylated horseradish peroxidase (1)– Alkaline phosphatase (1)– NBT/BCIP (1)
28
vWF Multimer AnalysisVisualization by Luminography
• Primary antibody source– Polyclonal rabbit anti-human vWF (1 – DAKO, 1 –
unknown vendor)– Goat anti-human vWF
• Secondary antibody source– Unknown animal source conjugated with horseradish
peroxidase (1 - Sigma)– Goat anti-rabbit IgG conjugated with alkaline
phosphatase (1- vendor unknown)– None listed (1)
29
vWF Multimer Analysis Visualization by Autoradiography
• Antibody source– In house pool of 9 monoclonal antibodies
labeled with 125Iodine using Iodogen method– Sheep anti-human vWF IgG (The Binding Site)
labeled with 125Iodine using chloramine T followed by affinity chromatography on vWF Sepharose
30
vWF Multimer Analysis Controls
Normal• Normal Pool Plasma (7)• Precision Biological NP
(2)• Daily Normal Control (1)• George King PNP (1)• George King Fact (1)
Abnormal• Abnormal Patient (4)• Type 2 Patient (4)• Humate-P (1)• BioData vWF (1)• Rarely Run (1)
Number of respondents performing assay
31
vWF Multimer Analysis
• Interpretation performed by– Laboratory scientist or physician (10)– Technologist (2)
• Result reporting– Image provided upon request (5 of 12 labs)
• Photocopy• Copy of nitrocellulose• Visual
– Would this be of value?• Yes (4)• Uncertain (1)• No (1)
32
Comments/Additional Questions
• Do most laboratoires doing FVIII Inhibitor testing use the Nijmegen method?
• Do most laboratories use lupus insensitive reagent for FVIII activity testing?
• Are abnormal results repeated on separate aliquot, if available?
• Effect of platelet source/type on RistocetinCofactor assay
• Develop “evidence based” guidelines for vWD testing including recommendations for ordering multimer analysis
33
Comments/Additional Questions
• What is the average turn-around-time for multimer analysis reports?
• What are criteria for distinguishing between Type 2A and 2B multimer patterns?
• Do you report a Type 2M based on multimersatellite patterns?
• Effect of sample age on discerning satellite banding?
34
vWF SurveysNASCOLA Results
NASCOLA ECAT NASCOLA ECAT
VWF Antigen (%) 44 + 7 45 120 + 13 123
VWF Ristocetin Cofactor (%) 33 + 10 32.7 88 + 18 86.1
FVIII Clot-based Activity (%) 32 + 6 36 99 + 12 101
Ratio: VWF:RCo/VWF:Ag 0.77 + 0.22 0.73 + 0.14
Ratio: FVIII/VWF:RCo 1.03 + 0.26 1.11 + 0.24
Ratio: FVIII/VWF:Ag 0.81 + 0.23 0.83 + 0.11
Survey 2004 - 01 (n=29) Survey 2004 - 02 (n=30)Test
NASCOLA values represent mean + SD
35
Relationship BetweenvWF Test Components
Survey 2004-01
• VWF:RCo/VWF:Ag Ratio <0.5– 2 labs (0.37, 0.45)
• FVIII/VWF:RCo Ratio > 2.0 or < 0.5– 1 lab (0.48)
• FVIII/VWF:Ag Ratio > 2.0 or <0.5– 1 lab (0.44)
Survey 2004-02
• VWF:RCo/VWF:Ag Ratio <0.5– 1 lab (0.44)
• FVIII/VWF:RCo Ratio > 2.0 or < 0.5– None
• FVIII/VWF:Ag Ratio > 2.0 or <0.5– None
36
Survey 2004 - 01 Survey 2004 - 02VWF Antigen Result = 46% Result = 123%
ECAT Vidas 8% (n=7) 9% (n=12)ECAT LIAtest 10% (n=85) 9% (n=64)ECAT Coamatic 16% (n=12) 12% (n=11)NASCOLA (all) 16% (n=28) 11% (n=31)
VWF Ristocetin Cofactor Result = 33% Result = 86%Dade Behring Automated 25% (n=64) 21% (n=72)Helena Aggregometry 24% (n=15) 31% (n=12)NASCOLA (all) 30% (n=29) 20% (n=30)
FVIII Clot-based Activity Result = 36% Result = 101%ECAT 20% (n-98) 16% (n=104)NASCOLA (all) 19% (n=28) 12% (n=30)
Interlaboratory Variability (% cv)Test / Method
Interlaboratory Coefficient of Variation
37
vWF: RCo Analysis by Methods
* 2004-01: One laboratory reported 0.45
Aggregometry (n=8)
Automated (n=10)
Others (n=14)
Aggregometry (n=8)
Automated (n=10)
Others (n=12)
NASCOLA Results 40 + 12 (cv=30%)
27 + 4 (cv=15%)
33 + 9 (cv=27%)
85 + 22 (cv=26%)
78 + 9 (cv=12%)
98 + 17 (cv=17%
ECAT Results 37 (cv=30%)
29 (cv=25%)
85 (cv=26%)
85 (cv=21%)
NASCOLA Ratio: VWF:RCo/VWF:Ag 0.60 + 0.08* 0.84 + 0.22* 0.64 + 0.06 0.77 + 0.16
NASCOLA Ratio: FVIII/VWF:RCo 1.14 + 0.22 1.00 + 0.27 1.30 + 0.20 1.04 + 0.20
Ristocetin Cofactor Assay
Survey 2004 - 01 Survey 2004 - 02
38
Effect of Independently Assayed Reference Plasma/Standard
Use Manufacture Assayed Value
(n-29)
Use Independent Assayed Value
(n=6)
Use Manufacture Assayed Value
(n-22)
Use Independent Assayed Value
(n=7)
VWF Antigen (%) 44 + 7 47 + 5 120 + 13 119 + 8
VWF Ristocetin Cofactor (%) 33 + 10 27 + 7 88 + 18 80 + 9
FVIII Clot-based Activity (%) 32 + 6 29 + 10 99 + 12 93 + 11Ratio VWF:RCo/VWF:Ag (<0.5)
0.77 + 0.22 (0.45)
0.65 + 0.05 (0.37)
0.73 + 0.14 (0.44) 0.67 + 0.07
Ratio FVIII/VWF:RCo (<0.5)
1.03 + 0.26 (0.48) 1.20 + 0.19 1.11 + 0.24 1.11 + 0.19
Ratio FVIII/VWF:Ag (<0.5)
0.81 + 0.23 (0.44) 0.83 + 0.11 0.83 + 0.11 0.80 + 0.13
Test
Survey 2004 - 01 Survey 2004 - 02
39
Summary
• NASCOLA performance on vWF surveys 2004-01 and 2004-02 are comparable to that of ECAT
• There does not appear to be a difference in results reported using manufactured assayed standards or independently assayed reference plasmas/standards
• Automated Ristocetin Cofactor assay appears to underestimate vWF activity
• Automated Ristocetin Cofactor assay does not appear to have a major effect on improving precision