2004 nascola annual scientific meeting

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

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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

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Questionnaire Distribution

• 39 surveys distributed by e-mailed• 34 completed surveys returned• Response rate: ~87%

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Goals /Aims

• Understand laboratory practices

• Understand test strategies

• Understand result interpretation

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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

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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

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vWF Antigen Method

• Automated: 86%– Latex Agglutination

• ELISA: 11%• Immunoelectrophoresis: 3%

Numbers represent percent respondents

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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%


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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


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vWF Activity Testing

• Ristocetin cofactor activity– Aggregometry: 51%– Automated: 42%– ELISA: 6%– One respondent does not perform vWF activity


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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%


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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%


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Ristocetin Concentration

• Reported concentration range:– 1.11ng/ml – 10 mg/ml

• 1.0 mg/ml: 18%• 1.5 mg/ml: 13%


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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

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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%


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FVIII Assay Buffer

• Owrens: 55%• Saline: 18%• IBS: 20%• IL Diluent: 6%• HEPES: 3%


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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

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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%


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FVIII ActivityInhibitor Effect Definition

• Delta 10% between dilutions (15%)



• >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

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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


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Collagen Binding Assay

• 19% of respondents perform collagen binding assay

• Performed in addition to Ristocetin Cofactor assay (some for research purposes only)

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vWF Multimer Analysis

• 52% of respondents send out samples for multimer analysis

• 35% of respondents perform vWF multimeranalysis on site

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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

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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

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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

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VWF Multimer AnalysisVisualization Methods

• Western Blot: 6 laboratories• Luminescence: 3 laboratories• Autoradiography: 2 laboratories• Unspecified: 1 laboratory

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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)

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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)

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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

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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

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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)

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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

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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?

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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

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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

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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

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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

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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

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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

2004 nascola annual scientific meeting

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