NASCOLA Annual Meeting
December 7, 2007
Hilton Atlanta Hotel
Atlanta, GA
MEETING AGENDA
Executive Secretary & Treasurer Report
NASCOLA Committee Reports
PT & EQA (Dr. Peerschke)
Education (Dr. Zehnder)
Website (Dr. Hayward)
Membership (Dr. Van Cott)
QA Program (Dr. Meijer)
“State of Association” (Dr. Hayward)
2007 Annual Report
NASCOLA Executive Secretary
Elizabeth Plumhoff
BS, MT(ASCP) CLS(NCA)
2007 Accomplishments and
Collaborations
 Act as Executive Committee resource – primary contact for
NASCOLA membership
 Prepare and distribute NASCOLA Newsletter (July 2007) with input
from Committee chairs and Executive Officers
 Participate in Subcommittee activities and conference calls
directed by Executive Committee
as
 Collaborate with Dr. Cathy Hayward and Dave Hayward on Website
design and processes
 Maintain accurate membership list and communicate Proficiency
Testing program participants to ECAT
Financial Summary
Dr. James Zehnder, Treasurer
Item
Amount
Beginning Balance January 1, 2007
$36,101
NASCOLA Dues & PT/EQA Program Institution Member Category
2007
$84,806
NASCOLA Dues &/or PT/EQA Program Associate Member
Category 2007
$14,000
NASCOLA Dues Individual Member Category 2007
$150.00
Payment to ECAT for 2007 PT/EQA Program
($65,123)
Other expenses
($26,934)
Balance (December 31, 2007) [ESTIMATED]
$43,000
NASCOLA Expenses 2007
Misc., $1,995
Executive Secretary, $6,750
Web SW, $9,000
Executive Secretary
OK CVA, $3,250
Web SW
OK CVA
NASCOLA-Mayo Diinner, $3,820
NASCOLA-Mayo
Diinner
ECAT
Misc.
ECAT , $65,123
Dollar vs. Euro
• Significant impact on
NASCOLA expenses
– ECAT payment is in
Euros
– Price unchanged for
2008
– Impact depends on
dollar value in 2008
Value of 1 Euro in USD
•
NASCOLA Committee
Reports & Updates
PT & EQA: Dr. Peerschke
Education: Dr. Zehnder
Website: Dr. Hayward
Membership: Dr. Van Cott
QA Program: Dr. Marlar
PT EQA
2007 Annual Report
Ellinor I. Peerschke, Chair
Working Groups
Von Willebrand’s Disease
• Wayne Chandler, M.D., Chair
• Ken Friedman, M.D.
• Donna D. Castellone, M.A., MT
(ASCP) SH
• Ellinor I. Peerschke, Ph.D.
FACTORS
• George Rodgers, M.D., Chair
• David Chance, M.T. (ASCP)
Thrombophilia Subcommittee
• Elizabeth Van Cott, M.D., Chair
• John Heit, M.D.
• Stephen Johnson, M.T.
• Rita Selby, M.D.
Lupus Anticoagulant Subcommittee
• Chuck Eby, M.D., Chair
• Agnes Aysola, M.D.
• Larry Brace, Ph.D.
• Joan Mattson, M.D.
• Kandice Kottke-Marchant, MD,
Ph.D
Marlies Ledford-Kraemer, MBA, BS, MT(ASCP)SH - ECAT liason
Activities
• Thrombophilia Testing
– Report on ATIII testing presented at Mayo-NASCOLA
meeting: Rita Selby
• VWF Multimer Analysis
– Report on Multimer Analysis presented at MayoNASCOLA meeting: Wayne Chandler
• Lupus Anticoagulant Testing
– Chuck Eby: new subcommittee chair
• Factor VIII Inhibitor Survey Results
– Overview presented at this meeting: Ellinor Peerschke,
Donna Castellone, Marlies Ledford-Kraemer
2008 Goals
• Develop guidelines for F VIII inhibitor
testing (Ellinor Peerschke)
• Develop questionnaire to better understand
interpretation and reporting of VWF test
results (Wayne Chandler) in North America
• Evaluate functional APCR testing by
NASCOLA laboratories (Betsy VanCott)
Education Committee
•
Dr. James Zehnder, Chair
NASCOLA Education
Committee Update
• HIT Survey - in press, Thrombosis and
Haemostasis
– ECAT Survey
– Proficiency testing
• HTRS/NASCOLA conference April 2008
• Mayo/NASCOLA conference 2009
• Pediatric Subcommittee - Anthony Chan
Website Committee Update
NASCOLA Annual Meeting
December 7, 2007
Catherine Hayward, MD, PhD
Website Committee Chair
Membership Committee
•
Dr. Elizabeth Van Cott, Chair
Membership Committee
Re-established December, 2006
Elizabeth Van Cott
CHAIRPERSON
• Lesley Black
• Donna Castellone
– Associate Member Liaison
• Cathy Hayward
• William Nichols
•
•
•
•
•
Ellinor Peerschke
Beth Plumhoff
Alvin Schmaier
Barbara Young
James Zehnder
Membership Status
2000
2001
2002
2003
2004
2005
2006
Paid
Members
13
18
28
37
53
70
75
Institution
Members
13
18
28
37
44
56
59
Associate
Members
0
0
0
0
9
12
14
Individual
Members
0
0
0
0
2
2
2
5
2
2
4
Honorary
Members
Member in
training
2007
80
61
14
5
Membership Status
Membership
90
80
Honorary Members
Members (number)
70
Individual Members
Associate Members
60
Institution Members
50
40
30
20
10
0
2000
2001
2002
2003
2004
2005
2006
2007
Every Year NASCOLA
Associate Members
Thank you!
•
•
•
•
•
•
Precision Biologic
Chrono-log Corporation
Dade Behring
Diagnostica Stago
DiaPharma
George King Bio-Medical
Membership Committee
Goals and Activities
• Recruitment of new associate members and
institutional members
– Invitation letters written by committee
– Special thank you to Cathy Hayward, Lesley
Black, Barbara Young and Betsy Van Cott for
contracting potential institutional members in 2007
– Special thank you to Donna Castellone for her role
as Associate Member Liaison
Membership Committee
Goals and Activities
• Recruitment, continued:
– “Top 10 Reasons to Join NASCOLA” assembled:
quotes from current members
http://nascola.org/Testimonials.aspx
Submit your own quote, if you want
Membership Committee
Goals and Activities
• Assist Executive Committee with databases
and financial analysis/decisions about
membership fees
• Communication to NASCOLA membership
– Newsletter to membership sent summer 2007
(compiled by Beth Plumhoff)
– Information sent in response to inquiries about
joining NASCOLA (Beth Plumhoff and others)
Membership Committee
Goals and Activities
• Develop new benefits of membership
– Associate Member Awards Program (requested by
Associate Members): Sponsorship of NASCOLA
institutional member dues and proficiency costs
(recipient/s chosen by Associate Member);
acknowledge Associate Member on website
– Members hosting a meeting can post
announcement on NASCOLA website
Membership Committee
Goals and Activities
• Develop new benefits of membership,
continued
– Associate Member Educational Programs:
Associate Members offered to sponsor
educational conferences with NASCOLA
– Members can have NASCOLA logo and
link on their website
Membership Committee
• Contact us with your ideas about increasing the
values of membership
• Feel free to tell others about the value of being part of
NASCOLA
• Feel free to submit a quote/comment/vignette for the
“Top 10 Reasons to Join NASCOLA” list
•
NASCOLA Quality
Assessment Program
Dr. Richard Marlar, Director
NASCOLA Quality Assessment
Program Highlights
• Total member participants = 62
– Complete Programs = 43
– Partial Programs = 19
NASCOLA Quality Assessment
Program Highlights
• New Modules for 2007
–Thrombin Generation Test
–HIT testing Pilot
NASCOLA Quality Assessment
Program Highlights
• New Modules for 2008
– HIT testing (1 survey / year)
– Molecular Modules (2 surveys / year)
• Module A ( Factor V Leiden (G1691A), Prothrombin
Gene Mutation (G20210A), Factor XIII (V34L), MTHFR (C677T and A1298C),
Glycoprotein IIIa (L33P), PAI-1 (4G/5G polymorphism), beta-Fibrinogen (G455A),
VKORC (Vitamin K Epoxide Reductase Complex) 1 (G1639A), FSAP (factor VII
activating protease) Marburg I (G1601A)
• Module B (Apo E, Apo B100, Alpha-1-Proteinase Inhibitor, ACE I/D, CETP)
• Module C (UGT-1A, TPMT, CYP2D6)
• Module D (HFE C282Y, HFE H63D, HFE S65C,Lactase-Phlorizin Hydrolase LPH
– Homocysteine (4 surveys / year)
– Factor XIII (Pilot program)
“State of the Association - 2007”
NASCOLA Annual Meeting
December 7, 2007
Catherine Hayward, MD, PhD
NASCOLA President 2007-2009
NASCOLA 2007 Highlights
•Elizabeth Plumhoff - Executive Secretary (3/06)
•Alvin Schmaier – Immediate Past President
•Membership Committee (Betsy Van Cott)
Many new initiatives, including “testimonials” on website
•Memberships as of November 2007
Institutional members 61 (from 59 as of 12/06)
Associate members 14
Individual members 5
*Honorary members 4
*Trainee memberships introduced - now 5
•Associate Membership Liaison (Donna Castellone)
*new!
NASCOLA 2007 Highlights
•PT Survey Program (Director: Richard Marlar)
•Continued collaboration with ECAT (Piet Meijer)
•New surveys piloted
•Expanded survey menu options for 2008
•ECAT Liaison (Marlies Ledford-Kraemer)
•Major NASCOLA Website Redesign
(Catherine & David Hayward)
•Migration to new site, introduction of many new features
NASCOLA 2007 Highlights
•On-line questionnaires and meetings on SurveyMonkey
(Karen Moffat)
•PT & EQA survey analysis (Ellinor Peerschke)
•Treasurer - Financial solvency (Jim Zehnder)
•NASCOLA Secretary (Dot Adcock)
•New Reporting Committee (Dot Adcock)
•Education Committee (Jim Zehnder)
More NASCOLA publications and studies!
NASCOLA-Mayo Conference April 26-28th, 2007
Education Liaison (Bill Nichols)
NASCOLA 2007 Highlights
• Other Initiatives
Discussions with College of American Pathologists
on potential collaborations
Discussions with Hemophilia Thrombosis Research
Society on educational meeting collaboration for 2008
Scientific Meeting
8:00 – 9:00 pm
Theme: “Update on NASCOLA Investigations, Publications and Study Plans”
Moderator: Dr. James Zehnder (Chair, Education Committee)
1.
2.
3.
4.
5.
6.
Ms. Marlies Ledford-Kramer, CLOT ED: Results of the first ECAT proficiency
challenge for HIT
Dr. James Zehnder, Stanford Medical Center: The NASCOLA survey on HIT testing
(now in press in Thrombosis and Haemostasis) and new data on how testing for HIT
differs between North America and Europe
Dr. Menaka Pai, University of Toronto Training Program in Adult Hematology:
Plans for a NASCOLA questionnaire on critical values for specialized coagulation
laboratories
Dr. Anthony Chan, Hospital for Sick Children: Plans for a NASCOLA research on
pediatric coagulation testing
Dr. Ellinor Peersche, Mount Sinai School of Medicine: Factor VIII Inhibitors:
Update on research by the PT-EQA Committee
Dr. Catherine Hayward, Hamilton Health Sciences-McMaster and Ms. Marlies
Ledford-Kramer, CLOT ED: Update on new initiatives to improve platelet function
testing with practice guidelines
Results of the first ECAT
proficiency challenge for HIT
Ms. Marlies Ledford-Kramer, CLOT-ED, Inc
NASCOLA/ECAT Liaison
ECAT Foundation
International EQA Programme in Thrombosis and Haemostasis
www.ecat.nl
Inter-laboratory comparison of
laboratory testing of Heparin Induced
Thrombocytopenia (HIT).
Preliminary results of a pilot study
Presented by:
Marlies Ledford-Kraemer
CLOT-ED, Inc
NASCOLA/ECAT Liaison
Piet Meijer, PhD
Director
ECAT Foundation
Leiden, The Netherlands
ECAT Foundation
International EQA Programme in Thrombosis and Haemostasis
Number of participants
:
Number of responders
:
www.ecat.nl
102
88 (86%)
Percentage responders NASCOLA:
83% (30/36)
Percentage responders ECAT :
88% (58/66)
Sample 1
:
HIT positive
Sample 2
:
Negative control
ECAT Foundation
International EQA Programme in Thrombosis and Haemostasis
www.ecat.nl
Methods
Immunologic
PAGIA (Diamed)
Asserachrom HPIA (Stago)
Anti-PF4 (GTI)
Zymutest IgG (Hyphen)
Zymutest IgM (Hyphen)
Unknown
ECAT
NASCOLA
33
21
8
2
1
1
7
18
5
18
2
1
1
2
2
-
Functional
Platelet aggregation
Platelet activation
Serotonin Release Assay
Flowcytometry
ECAT Foundation
International EQA Programme in Thrombosis and Haemostasis
www.ecat.nl
Results
SAMPLE-1
Immunologic
Functional
Negative
5
8
Positive
88
9
Borderline
4
1
Inconclusive
-
4
ECAT Foundation
International EQA Programme in Thrombosis and Haemostasis
www.ecat.nl
Results
SAMPLE-2
Immunologic
Functional
Negative
90
19
Positive
2
1
Borderline
3
-
Inconclusive
-
2
ECAT Foundation
International EQA Programme in Thrombosis and Haemostasis
Sample 1
www.ecat.nl
ECAT / NASCOLA
Immunologic
Pos
Neg
Border
PAGIA (Diamed)
Asserachrom HPIA (Stago)
Anti-PF4 (GTI)
Zymutest IgG (Hyphen)
Zymutest IgM (Hyphen)
Unknown
27 / 0
21 / 7
8 / 18
1/0
3/0
3/0
Inconcl
1/0
1/0
1/4
Functional
Platelet aggregation
Platelet activation
Serotonin Release Assay
Flowcytometry
7/0
1/0
0/1
4/2
0/1
1/0
1/0
2/0
1/0
1/0
ECAT Foundation
International EQA Programme in Thrombosis and Haemostasis
Sample 2
Immunologic
PAGIA (Diamed)
Asserachrom HPIA (Stago)
Anti-PF4 (GTI)
Zymutest IgG (Hyphen)
Zymutest IgM (Hyphen)
Unknown
www.ecat.nl
ECAT / NASCOLA
Pos
1/0
1/0
Neg
30 / 0
20 / 7
6 / 18
2/0
1/0
1/5
Border
Inconcl
2/0
1/0
Functional
Platelet aggregation
Platelet activation
Serotonin Release Assay
Flowcytometry
1/0
13 / 2
1/0
1/2
1/0
1/0
1/0
ECAT Foundation
International EQA Programme in Thrombosis and Haemostasis
www.ecat.nl
Conclusion
•
•
•
•
First large inter-laboratory comparison on HIT testing
About 80% of the performed assays are immunological tests
In Europe, the PGIA from Diamed is the most frequently used test
In North-America, the anti-PF4 from GTI is the most frequently
used test
• Sample 1 is correctly identified as HIT positive by 91% of the
users of an immunological test and 41% of the users of a
functional test
– Misclassification of Sample 1 with an immunological test is only
caused by the PGIA (Diamed) and Zymutest (Hyphen)
• Sample 2 is correctly identified as HIT negative by 95% of the
users of an immunological test and 86% of the users of a
functional test
– Misclassification of Sample 2 with an immunological test is only
caused by the PGIA (Diamed), HPIA (Stago), and anti-PF4 (GTI)
The NASCOLA survey on HIT
testing and new data on how testing
for HIT differs between North
America and Europe
Dr. James Zehnder
Stanford Medical Center
NASCOLA Treasurer
NASCOLA Education Committee Chair
HIT Practices Survey: ECAT
Results
Jim Zehnder, M.D.
Stanford University
December 7, 2007
Heparin-Induced Thrombocytopenia
• Serious complication of heparin therapy
• Clinico-pathologic entity
• Laboratory practices have important role in
diagnosing or excluding HIT
HIT Testing Methodologies
• PF-4/Heparin Antibodies (Antigen assays)
– Rapid tests: PaGIA (DiaMed/Ortho), PIFA (Akers
Biosciences); (?NOT AVAILABLE IN N. AMERICA)
– ELISA (GTI, Diagnostica Stago)
• Platelet Activation Assays
– Serotonin release assay (SRA)
– Heparin induced platelet aggregation (HIPA)
– Washed platelets vs platelet-rich plasma
Survey
• 67 questions related to laboratory testing for HIT
• Distributed to:
– NASCOLA members: 44/59 (79%) responded by
XXXXX
– ECAT (European Concerted Action on Thrombosis)
members: 42/__ (--) responded by Sept 15 2007
– Number of laboratories that responded to individual
questions varied
HIT Testing and Pre-Analytical Variables
ECAT
NASCOLA
Number laboratories responded to
survey
42
44
Perform HIT testing
40/41 (98%)
44/44 (100%)
Median # of tests per year (range)
61 (0-800)
950 (55-7500)
Require clinical information for
testing
33/42 (79%)
15/43 (35%)
Require approval for testing
20/41 (49%)
8/42 (19%)
Have sample requirements
25/40 (63%)
25/40 (63%)
? Availability of testing)
Pre-Analytical Variables: Concerns
ECAT
NASCOLA
Concerns about pre-analytical
variables and HIT testing
18/32 (56%)
32/40 (80%)
Concerned heparin could be in
sample
13/16 (81%)
29/37 (78%)
Sample may have been drawn too
early
12/19 (63%)
27/36 (75%)
HIT: Antigen Assays
ECAT
NASCOLA
Test for PF4/heparin antibodies
31/32 (97%)
35/40 (80%)
Rapid: PaGIA
22/33 (67%)
0/34 (0%)
Rapid: PIFA
0/33 (0%)
0/34 (0%)
PF4/Polyanion ELISA (GTI)
4/33 (12%)
24/34 (71%)
PF4/Heparin ELISA (Stago)
13/33 (39%)
9/34 (27%)
Detect and/or report antibody
subclass
2/27 (7%)
5/38 (13%)
Problems performing/ interpreting
assays
14/31 (45%)
14/25 (56%)
HIT Antigen Testing: Reporting
Report
ECAT
NASCOLA
Positive results
24/26 (92%)
30/38 (79%)
Negative results
23/26 (89%)
30/38 (79%)
Borderline results
4/26 (15%)
12/38 (32%)
Indeterminate
results
8/38 (21%)
1/26 (4%)
Interpretive Comments for Antigen Testing
Results: ECAT Survey
• “The sensitivity of… the assay may be increased by
repeating 4 to 10 days after any heparin exposure”
• “Negative ELISA rules out HIT diagnosis with 98%
security”
• “A negative test does not exclude the diagnosis of HIT”
• “HIT is a clinicopathologic syndrome, please correlate
with clinical presentation and platelet count”
• We propose treatment and laboratory monitoring.
HIT: Platelet Activation Assays
ECAT
NASCOLA
Perform platelet activation assays,
including:
16/33 (49%)
14/39 (36%)
SRA with WP
0/16 (0%)
6/14 (43%)
SRA with PRP
1/16 (6%)
1/14 (7%)
Heparin induced platelet aggregation
(HIPA) with PRP
12/16 (75%)
6/14 (43%)
Heparin induced platelet activation by
flow cytometry
1/16 (6%): PRP
1/14 (7%): WP
HIPA, microplate method, with WP
2/16 (13%)
0/14 (0%)
HIPA, aggregometry, with WP
1/16 (6%)
0/14 (0%)
Platelet donors prescreened for HIT
reactivity
5/14 (36%)
10/11 (91%)
HIT Activation Assay Practices:
Positive Controls
Use positive control
ECAT
NASCOLA
11/16 (69%)
14/14 (100%)
5/16 (31%)
10/14 (71%)
Including:
Previously tested, strongly positive
pt sample
Previously tested, weakly positive pt 4/16 (25%)
sample
4/14 (29%)
Platelet activating agonist control
4/14 (29%)
5/16 (31%)
HIT Activation Assay Practices:
Negative Controls
ECAT
NASCOLA
7/16 (44%)
13/14 (93%)
Healthy control
3/16 (19%)
7/14 (50%)
Previously tested negative patient
sample
2/16 (13%)
4/14 (29%)
Saline Control
0/16 (0%)
1/14 (7%)
Use negative control
Including:
Activation Assay: Reporting Practices
Report:
ECAT
NASCOLA
Positive results
16/16 (100%)
12/14 (86%)
Negative results
16/16 (100%)
12/14 (86%)
Weak positive results
1/16 (6%)
4/14 (29%)
Borderline results
3/16 (19%)
4/14 (29%)
Indeterminate results
3/16 (19%)
10/14 (71%)
Nonspecific results
4/16 (25%)
5/14 (36%)
Provide interpretive comment 16/16 (100%)
10/14 (71%)
HIT Testing: Comments and
Concerns (ECAT)
• “Are guidelines available for laboratory testing i.e.
sequence of testing, heparin in sample, etc”
• “The problem is that the test is not frequently requested,
until a [seminar] is done, then we observe a sudden rise in
the number of tests, that are often not appropriately
requested”
• “I think that HIT testing should be performed in
laboratories with a close cooperation with haematologists”
• “The majority of negative results don’t give information”
Summary: HIT Survey Results
• Variability between and within ECAT and NASCOLA
survey groups:
– Volume of testing
– Clinical background/ Approval requirements for testing
– HIT antigen testing methodologies
• Use of rapid immunoassays vs ELISA
– HIT functional assay methodologies
• SRA vs HIPA
• Washed platelets vs. platelet rich plasma
• Use of controls
Summary, Continued
• Variability in testing has important clinical
consequences
• Many laboratories report problems or concerns
related to testing
• Supports finding of NASCOLA survey for need
for proficiency testing to drive international
consensus regarding HIT testing
Plans for a NASCOLA questionnaire
on critical values for specialized
coagulation laboratories
Dr. Menaka Pai
University of Toronto Training Program in
Adult Hematology
CRITICAL VALUES IN THE
COAGULATION LAB:
A PROPOSED NASCOLA MEMBER
SURVEY
By Menaka Pai, BSc MD FRCPC
Chief Hematology Resident, University of Toronto
What is a Critical Value?



First defined by George Lundberg in 1972 at the
Los Angeles County USC Medical Center
“A pathophysiologic state at such variance with
normal as to be life-threatening unless something
is done promptly and for which corrective action
can be taken.”
Lundberg GD. When to panic over abnormal values. Med Lab Obs. 1972;4:47-54.
Critical Values and the
Health Care Provider


Health care providers are directly responsible for
acting on abnormal laboratory results
This is increasingly difficult:
 We
care for large numbers of patients we do not know
personally
 We work in both inpatient and outpatient settings
 We work during and after regular work hours
 We are unfamiliar with the “normal” and “critical”
ranges of many of the tests we order!
Critical Values and the Laboratory

Laboratory staff are…
 Often
more aware of the significance of abnormal
laboratory results
 Uniquely placed to efficiently communicate results to
clinicians
 Can work with clinicians to improve patient outcomes

Critical values are widely used and medically
important, but they are handled differently from
laboratory to laboratory
Interest in Critical Values Is High

Have been incorporated into…

Practice parameters:


Federal regulations:


Clinical Laboratory Improvement Amendments Act (1988)
Standards of regulatory agencies:


American Society of Clinical Pathology’s practice parameters
(1997)
Joint Commission on Accreditation of Healthcare Organizations’
National Patient Safety Goal (2006)
Have been the subject of national and institutional
quality improvement studies and surveys
The Role of Critical Values in the
Coagulation Laboratory

Recent review of the literature found…
 Several
publications discussing critical values in
biochemistry, microbiology and hematology
laboratories
 No publications discussing critical values in coagulation
laboratories!

We would like to find out what YOU think about
critical values!
Our Proposal

Survey members of the North American Special
Coagulation Laboratory Association (NASCOLA) in
order to…
 Establish
benchmark / consensus data on critical value
ranges for commonly performed special coagulation
tests, and any modifications for special populations
 Determine the current pattern of practice for reporting
critical values in North American special coagulation
laboratories
 Determine how laboratories design their critical value
policies
The Survey



Online (on Survey Monkey)
All responses will be confidential
Areas of interest:
Your critical range / value for specific coagulation tests
 Your thoughts on the need for different critical values for
special populations (ie. Neonates, obstetrical patients)
 How your laboratory reports inpatient and outpatient
critical values
 How your laboratory’s critical value policy was developed

The Next Steps



Elicit feedback from NASCOLA members
Develop Critical Values Survey and launch it in the
new year
Determine if Critical Values should be a focus of
further study…
A
followup survey on critical value reporting?
 A NASCOLA working group to address critical values in
special coagulation?
Thank you for considering this initiative!
And thank you to…
Dr. Catherine Hayward
Ms. Karen Moffat
Dr. Dot Adcock
Please contact me with further questions or concerns! menakapai@hotmail.com
Plans for a NASCOLA research on
pediatric coagulation testing
Dr.Anthony Chan
Hospital for Sick Children
Factor VIII Inhibitors: Update
on research by the PT-EQA Committee
Dr. Ellinor Peerschke
Mount Sinai Medical Center
NASCOLA PT/EQA Committee Chair
Performance Summary:
Factor VIII Inhibitor Testing
NASCOLA PT/EQA
Ellinor I. Peerschke
Donna D. Castellone
Marlies Ledford-Kraemer
2007
84
ECAT: FVIII Inhibitor
Proficiency Testing Challenge

Laboratory Participation ECAT and
NASCOLA




March 2005 Pilot (135 responses)
June 2006 (104 responses)
January 2007 (109 responses)
The largest FVIII inhibitor proficiency testing
challenge conducted in North America

32-35 NASCOLA laboratories participated
Purpose



Investigate methods used in clinical
laboratories for FVIII inhibitor quantification
Examine between laboratory variation
Identify possible determinants for interlaboratory variation
All Results: Pilot Survey 2005

Sample A





Sample B





FVIII deficient plasma enriched with plasma from a high titer positive
FVIII inhibitor plasma resulting in a titer of 15-20 BU/ml
14.9 BU/ ml (Range 2-67 BU/ml)
Most frequent replies (10,15, 20 BU/ml)
CV 56%
FVIII deficient plasma enriched with plasma from a high titer positive
FVIII inhibitor plasma resulting in a titer of 1.0-1.5 BU/ml
1.86 BU/ ml (Range 0-42 BU/ml)
Most frequent replies (1.0, 1.5, 2.0 BU/ml)
CV 202%
Sample C



FVIII deficient plasma
120 of 127 labs reported inhibitor levels below detection limit
7 labs reported inhibitor titers of 0.05, 0.1, 0.5, 2.9, 2.0, 8.0
All Results: 2006 Survey

Sample 06.31





Low titer FVIII inhibitor: ~1.5 BU/ml
All results: mean 1.29 BU/ml (Range 0.3 – 3.6 BU/ml)
Most frequent replies (1.0, 1.5, 0.5-2.0)
CV 42%
Sample 06.32




Negative Control
Range 0- 6.4 BU/ml
Most frequent replies (0, 0.5 BU/ml)
Lower limit of detection (<0.5 BU/ml, some labs reported
0.75, 1.0, 1.5, 2.0 BU/ml)
NASCOLA Results (2006)
Result Distribution (Sample 06.32)
20
40
15
30
# Labs
# Labs
Result Distribution (Sample 06.31)
10
20
5
10
0
0
0-0.5
0.6 -1.0
1.10-1.5
1.6-2.0
2.1-2.5
Bethesda Titer
Low inhibitor sample ~1.5 BU/ml
NASCOLA mean 1.3 BU, CV 31%
(ECAT mean 1.29 BU)
0
0.1-0.5
Bethesda Titer
Negative Control
All Results: 2007 Survey

Sample 06.63




Negative control
Most labs reported BU below detection limit or 0 BU/ml
Highest reported result 2.2 BU/ml
Sample 06.64




Low titer inhibitor (1.5 – 2.0 BU/ml)
1.69 BU/ml (Range 0.4 – 6.1 BU/ml)
Most frequent replies (1.0, 1.5, followed by 0.5 and 2.0)
CV 51.5%
NASCOLA Results (2007)
Result Distribution (Sample 06.63)
Result Distribution (Sample 06.64)
12
8
6
# Labs
# Labs
10
4
2
0
0-0.5 0.6 1.0
1.11.5
1.62.0
2.12.5
2.63.0
3.13.5
3.64.0
Bethesda Titer
Low inhibitor sample: 1.5-2.0 BU/ml
NASCOLA 1.9 BU, CV 39%
(ECAT 1.69 BU)
35
30
25
20
15
10
5
0
0
<0.5
Bethesda Titer
Negative Control
Reported Limits of Detection by
NASCOLA Laboratories
18
# Labs
16
14
12
10
8
6
4
2
0
0
0-0.5
0.6-1.0
Bethesda Titer
>1.0
General Observations:
All laboratories



Significant variation among laboratories,
although reasonable consistency among
many laboratories
Coefficient of variation is better for Nijmegan
method than for Bethesda method
Almost ¾ of all participants perform the
Bethesda assay
NASCOLA Laboratories
F VIII Inhibitor Assay Conditions



85% perform Bethesda assay
94% perform 1+1 normal plasma/patient plasma incubation
78% use buffered normal plasma
 65% commercial source


12% homemade
42% use normal plasma calibrated against WHO standard
7 different sources of commercial FVIII deficient plasma
Diluent
 42 % use Immidazole buffer
 16% FVIII deficient plasma
 3% albumin
 29% other




80% Precision Biologic
20% George King
FVIII Level of Normal Plasma
# Labs
Remaining FVIII in Normal Plasma Control
16
14
12
10
8
6
4
2
0
20-30
(Outliers 100,125%)
31-40
41-50
F VIII (%)
FVIII level of Normal Plasma = 76 – 140%
51-55
Sources of Variation

Methodological variation (affecting stability of FVIII)




Buffered vs unbuffered plasma
FVIII deficient plasma vs immidazole buffer
Patient plasma dilution
Variations affecting result interpretation




Variability in FVIII assay
FVIII level in normal pool plasma
Definition of FVIII inhibitor assay detection limit
BU conversion chart (inconsistencies)
Source of Variability: Bethesda Unit
Conversion



We found 3 different charts for converting
residual F VIII activity to BU
Some charts read from 75%-25% residual F
VIII activity, while others read from 100%25% or 100%-6% residual F VIII
Reading from one chart may give a negative
result (<0.5 BU), while others may provide a
false positive result, which will be further
affected by multiplication by the relevant
dilution factor
Comparison of Bethesda Unit Conversion
Tables
Residual
F VIII
75%
BU - #1 BU - #2
BU - #3
0.40
0.39
0.50
70%
0.50
0.50
0.60
60%
0.725
0.72
0.80
55%
0.85
0.86
0.90
30%
1.70
1.75
1.80
27%
1.85
1.90
1.89
PT/EQA Actions


Develop consensus guidelines for FVIII
inhibitor detection and quantification
Consider





Buffer for dilution and F VIII stabilization
F VIII source
Assay detection limit
Standard BU conversion chart
Standardize F VIII assay
Update on new initiatives to improve
platelet function testing with practice
guidelines
Dr. Catherine Hayward
Hamilton Health Science-McMaster
NASCOLA President, Website Committee Chair
Ms. Marlies Ledford-Kramer, CLOT-ED, Inc
NASCOLA/ECAT Liaison
Standardization of Platelet Function Testing
CLSI Guideline H58-P
Platelet Function Testing by Aggregometry
Marlies Ledford-Kraemer
MBA, BS, MT(ASCP)SH
CLOT-ED, Inc
Conflict of Interest
• None to disclose
Clinical and Laboratory
Standards Institute (CLSI)
• Global, nonprofit, standards-developing organization
that promotes the development and use of voluntary
consensus standards and guidelines within the
healthcare community
• Based on the principle that consensus is an efficient
and cost-effective way to improve patient testing and
services
• Committee members for the development of
consensus guidelines consist of individuals from
industry, academia, and government
Members:
Subcommittee on Platelet Function Testing
(Area Committee on Hematology)
•
•
•
•
•
•
•
•
•
•
Douglas Christie, PhD: Dade Behring - Chairman
Leonthena Carrington, MBA, MT(ASCP): FDA Center for Devices
Eli Cohen, PhD: Haemoscope Corporation
Paul Harrison, PhD: Churchill Hospital, Oxford, UK
Thomas Kickler, MD: Johns Hopkins University
Marlies Ledford-Kraemer, MBA, MT(ASCP)SH: CLOT-ED, Inc.
Kandice Kottke-Marchant, MD, PhD: Cleveland Clinic
Alvin Schmaier, MD: Case Western Reserve University
Melanie McCabe White: Univ of Tennessee Health Science Cntr
David Sterry, MT(ASCP): CLSI Staff Liaison
Advisors to Subcommittee
•
•
•
•
•
•
•
•
Kenneth Ault, MD: Maine Medical Center
Thrity Avari, MS: Chronolog Corporation
Barbara DeBiase: Sienco, Inc.
Richard Marlar, PhD: Oklahoma City VA Medical Center
Margaret Rand, PhD: The Hospital for Sick Children, Toronto
Ravindra Sarode, MD: UT Southwestern Medical Center
Jun Teruya, MD: Baylor College of Medicine
William Trolio: Bio/Data Corporation
Timeline for H58
Proposed Guideline
• January 2004: project proposal submitted to CLSI by Dr Christie
– Rationale: Currently there are no specific standards or guidelines for clinical
laboratory platelet function testing….this proposal is intended to be a first step
toward providing for [that] standardization…
• August 2005: project approved
– Nominations for subcommittee membership submitted and approved
• May 2006: first conference call to determine writing assignments
• October 2006: two day on-site meeting at CLSI headquarters in
Pennsylvania to review first draft of document
• December 2006-April 2007: conference calls (5) to reach consensus
• April 2007: vote by subcommittee to accept consensus document
• June 2007: document submitted to Area Committee on Hematology for
review and vote
• June 30, 2007: proposed guideline published
• October 2007: review delegate comments and consider document revisions
Scope of H58-P Guideline
• Specifies requirements/recommendations for specimen collection,
pre-examination considerations, patient preparation, sample
processing, testing, and quality control in relation to platelet
function testing by aggregometry
• Covers anticoagulants, specimen storage and transport temperatures,
sample selection for various methodologies, establishment of
reference intervals, result reporting, assay validation, and
troubleshooting
• Intended for use by clinicians, hospital and reference laboratorians,
manufacturers, and regulatory agencies
• Guideline is not intended for use with global hemostasis, platelet
counting, flow cytometry, point-of-care, or research systems
• Guideline does not address therapeutic guidance or interpretation
Guideline Chapters and
Writing Assignments
• Introduction
– Paul Harrison
• Specimen collection and processing
– Marlies Ledford-Kraemer
• Light transmission aggregometry using platelet rich plasma (PRP)
– Alvin Schmaier and Melanie White
• Impedance aggregometry using whole blood
– Thrity Avari and Lee Carrington
• High shear device (platelet function analyzer)
– Doug Christie
• Quality control
– Tom Kickler and Barb DeBiase
Recommendations:
Pre-examination Issues
•
•
•
Knowledge of patient medication and dietary history
Evacuated tube systems, syringes, or butterfly cannulae systems may be used with
needle gauge sizes between 19 and 21 (23 gauge size acceptable for pediatrics)
Anticoagulant is 3.2 % (105–109 mmol/L) trisodium citrate
– Anticoagulant to blood ratio is 1 part trisodium citrate to 9 parts blood
• Suggest citrate be adjusted to compensate for hematocrits greater than 45%
•
•
Specimens should be transported at room temperature (20-25oC), hand carried (no
pneumatic tubes), and not be exposed to vibration, shaking or agitation
PRP sample preparation for light transmission aggregometry (LTA)
–
–
–
–
Centrifuge at 170g for 15 minutes at room temperature
Store PRP in a capped plastic tube with limited surface area-to-volume ratio
Platelet count of platelet poor plasma (PPP) should be less than 10 x 109/L
For PRP platelet counts in excess of 400 x 109/L, recommended target PRP count,
subsequent to dilution with autologous PPP, is 200-250 x 109/L
– Maintain PRP at room temperature and use within 4 hours after platelet donation
Recommendations:
Light Transmission Aggregometry
• LTA performance requires a standardized cuvette volume for PRP, stirring,
standardized light transmittance between 0 to 100%, a run time between 5-10
minutes, and a chart speed of > 2 cm/min
• Agonists
–
–
–
–
–
ADP: use at 0.5-10 μM final concentrations (typical starting concentration = 5μM)
Arachidonic Acid: use at a single dose between 0.5-1.6 mM
Collagen (type unspecified): use at 1-5 μg/ml (typical starting concentration = 2.0 μg/ml)
Epinephrine: use at a range of 0.5-10 μM (typical starting concentration = 5 μM)
Ristocetin (ristocetin induced platelet aggregation or RIPA):
• High dose: 0.8 – 1.5 mg/ml
• Low dose: < 0.5 mg/ml
– Gamma-thrombin: use at various concentrations (titrated)
• LTA reporting
– Percent final aggregation, percent maximum aggregation, slope (initial rate of chart
deflection), or in the case of ADP and epinephrine, the minimal concentration needed to
induce second wave aggregation
Recommendations:
Impedance Aggregometry
•
Whole blood platelet counts between 100 – 1,000 x 109/L are diluted with equal
parts preservative-free sterile saline immediately prior to testing
– Platelet counts less than 100 x 109/L are tested undiluted
•
Agonists
–
–
–
–
–
ADP: use at 5-20 µM
Arachidonic Acid: use at 0.5-1.0 mM
Collagen: use at 1-5 µg/ml
Epinephrine: not recommended as an agonist as fewer than 50% of subjects respond
Ristocetin (RIPA):
• High dose: 1.0 mg/ml
• Low dose: 0.25 mg/ml
•
•
ATP release is quantified by comparing the resulting luminescence values from
each test/agonist to a previously tested 2.0 nmole ATP standard
Reporting impedance aggregation without/with luminescence
– Amplitude, slope (maximum rate of chart deflection), ATP release, lag time, and area
under the curve (AUC).
Recommendations:
Quality Control
•
•
•
•
Simultaneously test a normal sample at the time a patient or patients is/are tested
When receiving a new shipment of reagent, determine its reactivity with normal
platelets
Participate in proficiency testing (exchange of samples)
Reference intervals need to be established for aggregation and secretion responses
for each agonist at specific concentrations that are used for patient testing
– Reference intervals must be reported with patient results
•
•
•
•
•
Measure precision by testing a minimum of ten samples in duplicate and calculating
the coefficient of variation of the duplicate pairs
Determine potency of an agonist by doing dose response to varying concentrations
of agonist
Perform independent temperature measurement using an appropriate probe in a
cuvette containing water or saline (and stir bar) inserted in a testing well
Mechanical stirrers need to have rpms validated at recommended intervals
If a laboratory has more than one platelet aggregometer, reproducibility between
instruments and even between channels on the same instrument must be validated
Selected References
•
•
•
•
•
•
Jennings LK, White MM. Platelet Aggregation. In: Michelson AD, Ed. Platelets 2nd
Edition. Amsterdam: Elsevier. 2007, 495-518.
Moffat KA, Ledford-Kraemer MR, Nichols WL, Hayward CP. Variability in clinical
laboratory practice in testing for disorders of platelet function: results of two surveys
of the North American Specialized Coagulation Laboratory Association. Thromb
Haemost 2005;93:549-53.
Zhou L, Schmaier AH. Platelet aggregation testing in platelet-rich plasma: description
of procedures with the aim to develop standards in the field. Am J Clin Path
2005;123:172-83.
Zucker MB. Platelet aggregation measured by the photometric method. Methods
Enzymol 1989;169:117-33.
Ingerman-Wojenski CM, Silver MJ. A quick method for screening platelet
dysfunctions using the whole blood lumi-aggregometer. Thromb Haemost
1984;51:154-6.
Kundu SK, Heilmann EJ, Sio R, Garcia C, Ostgaard RA. Characterization of an in
vitro platelet function analyzer - PFA-100. Clin Appl Thromb Hemost 1996;2:241-49.
ISTH 2007 Survey
Clinical Laboratory Responses
• Clinical laboratory responses = 245
• NASCOLA respondents = 43 (18% of total)
Aggregation tests performed annually
range: 3 to 5000 tests
(n=206 labs)
12%
Range of LTA
>500
401-500
3%
301-400
4%
14%
201-300
151-200
4%
126-150
8%
101-125
76-100
5%
7%
51-75
6%
37%
<= 50
0
5
10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
Number of LTA ran / year within range
Type of Aggregometry
n=121 responses
10%
31%
59% (n=145/245) collected samples into 3.2% sodium
citrate (n=98) or buffered sodium citrate (n=47)
51%
WBA not done
LTA not done
Do both but LTA more
frequent
Do both but WBA more
frequent
8%
Pre-analytical Variables
Patients are required to……
Abstain from
caffeine
n=26/202
Refrain from
smoking
n=25/206
Rest
n=53/201
Fast
n=38/162
0
10
20
30
40
50
60
70
80
90
100
Needles Used For Sample Collection
(n=179)
n=2
n=49
16 guage
19 guage
20 guage
21 guage
n=101
n=27
•32% (n=79/245) used a butterfly needle
Study a normal control in
parallel with the patient
No 49%
(n=99 / 204)
Yes 51%
(n=105 / 204)
Value of Adjusted Counts
n=119 responses
PRP Count Adjustment
n=173 responses
16%
15%
30%
39%
No Adjustment
45%
149 adjust with PRP
Only when plt count
exceeds limit
all samples adjusted
55%
200 x 10^9/L
250 x 10^9/L
300 x 10^9/L
Aggregation and Secretion
Studies Performed Together?
n=153 responses
Yes, on all patients
n=24
Yes, but only on
selected patients
n=29
Not performed
together
n=100
0
10
20
30
40
50
60
70
80
90
100
Maximal Time Allowed for Completing LTA
n=140 responses
Within 1 hour
Within 2 hours
Within 3 hours
Within 4 hours
Within 5 hours
Within 6 hours
0
10
20
30
40
50
60
percentage
70
80
90
100
Common LTA Agonists
at least one concentration
n=149 responses
high dose Ristocetin
low dose Ristocetin
ADP
Collagen
Epinephrine
Arachidonic Acid
U46619
TRAP
gamma-thrombin
PAF
0
10
20
30
40
50
60
70
80
90
100
Times for Monitoring
LTA Responses
Agonist (n)
ADP
Range (minutes)
3 – 10
Most common
5 minutes
Epinephrine
Collagen
TRAP
3 - 10
3 - 10
3 - 10
PAF
U46619
3 – 10
3 – 10
5 minutes (n=6)
10 minutes (n=6)
10 minutes
5 minutes
Arachidonic Acid
Gamma-thrombin
Ristocetin
3 – 10
3 - 10
3 – 10
5 minutes
10 minutes
5 minutes
Statistical Approach for
Determining LTA RI
Special statistical tests that allow for use of repeat measures on
some controls
Statistics using one value for each control
Mean plus or minus 2 standard deviations, with log transformation
of some data to normalize the distribution
Nonparametric statistical tests
Mean plus or minus 2 standard deviations
Not applicable - reference ranges not determined
0
5
10 15 20 25 30 35 40 45 50
Percentage
Note scale
Parameters Evaluated for LTA
amplitude of % agg at end of test
measure of lag phase
presence of a shape change
slope of aggregation
visual inspection of tracing
deaggregation
presence of "secondary wave"
maximal amplitude or % agg
0
10
20
30
40
50
60
Percentage
70
80
90 100
Parameters Included in Report
Amplitude of aggregation at the end of the observation
Presence of a shape change
Measure of lag phase
Slope of aggregation
Visual inspection of the tracing
deaggregation
Presence of "secondary wave"
maximal amplitude or % aggregation
Overall interpretative comment
0
10
20
30
40
50
60
Percentage
70
80
90
100
•