NASCOLA Annual Meeting December 7 2007 Hilton Atlanta

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

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 Mayo. NASCOLA 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 Van. Cott)

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, Ph. D 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 9 12 14 Individual Members 0 0 2 2 2 5 2 2 4 Honorary Members Member in training 2007 80 61 14 5

Membership Status

Every Year NASCOLA Associate Members Thank you! • • • Precision Biologic Chrono-log Corporation Dade Behring Diagnostica Stago Dia. Pharma 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 (G 1691 A), Prothrombin Gene Mutation (G 20210 A), Factor XIII (V 34 L), MTHFR (C 677 T and A 1298 C), Glycoprotein IIIa (L 33 P), PAI-1 (4 G/5 G polymorphism), beta-Fibrinogen (G 455 A), VKORC (Vitamin K Epoxide Reductase Complex) 1 (G 1639 A), FSAP (factor VII activating protease) Marburg I (G 1601 A) • Module B (Apo E, Apo B 100, Alpha-1 -Proteinase Inhibitor, ACE I/D, CETP) • Module C (UGT-1 A, TPMT, CYP 2 D 6) • Module D (HFE C 282 Y, HFE H 63 D, HFE S 65 C, 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, Ph. D 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 Survey. Monkey (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 -28 th, 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-Mc. Master 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, Ph. D 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-PF 4 (GTI) Zymutest Ig. G (Hyphen) Zymutest Ig. M (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-PF 4 (GTI) Zymutest Ig. G (Hyphen) Zymutest Ig. M (Hyphen) Unknown 27 / 0 21 / 7 8 / 18 1/0 3/0 Inconcl 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 2/0 1/0

ECAT Foundation International EQA Programme in Thrombosis and Haemostasis Sample 2 Immunologic PAGIA (Diamed) Asserachrom HPIA (Stago) Anti-PF 4 (GTI) Zymutest Ig. G (Hyphen) Zymutest Ig. M (Hyphen) Unknown www. ecat. nl ECAT / NASCOLA Pos 1/0 Neg 30 / 0 20 / 7 6 / 18 2/0 1/5 Border Inconcl 2/0 1/0 Functional Platelet aggregation Platelet activation Serotonin Release Assay Flowcytometry 1/0 13 / 2 1/0 1/0 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-PF 4 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-PF 4 (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: Pa. GIA (Dia. Med/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%) ? 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 PF 4/heparin antibodies 31/32 (97%) 35/40 (80%) Rapid: Pa. GIA 22/33 (67%) 0/34 (0%) Rapid: PIFA 0/33 (0%) 0/34 (0%) PF 4/Polyanion ELISA (GTI) 4/33 (12%) 24/34 (71%) PF 4/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 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 q q q March 2005 Pilot (135 responses) June 2006 (104 responses) January 2007 (109 responses) The largest FVIII inhibitor proficiency testing challenge conducted in North America q 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 q q Sample B q q 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 q q q 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 q q 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 q q 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) Low inhibitor sample ~1. 5 BU/ml NASCOLA mean 1. 3 BU, CV 31% (ECAT mean 1. 29 BU) Negative Control

All Results: 2007 Survey Sample 06. 63 q q q Negative control Most labs reported BU below detection limit or 0 BU/ml Highest reported result 2. 2 BU/ml Sample 06. 64 q q 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) Low inhibitor sample: 1. 5 -2. 0 BU/ml NASCOLA 1. 9 BU, CV 39% (ECAT 1. 69 BU) Negative Control

Reported Limits of Detection by NASCOLA Laboratories

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 q 65% commercial source 12% homemade 42% use normal plasma calibrated against WHO standard 7 different sources of commercial FVIII deficient plasma Diluent q 42 % use Immidazole buffer q 16% FVIII deficient plasma q 3% albumin q 29% other q 80% Precision Biologic 20% George King

FVIII Level of Normal Plasma (Outliers 100, 125%) FVIII level of Normal Plasma = 76 – 140%

Sources of Variation Methodological variation (affecting stability of FVIII) q q q Buffered vs unbuffered plasma FVIII deficient plasma vs immidazole buffer Patient plasma dilution Variations affecting result interpretation q q 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. 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 q q q 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-Mc. Master NASCOLA President, Website Committee Chair Ms. Marlies Ledford-Kramer, CLOT-ED, Inc NASCOLA/ECAT Liaison

Standardization of Platelet Function Testing CLSI Guideline H 58 -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, Ph. D: Dade Behring - Chairman Leonthena Carrington, MBA, MT(ASCP): FDA Center for Devices Eli Cohen, Ph. D: Haemoscope Corporation Paul Harrison, Ph. D: Churchill Hospital, Oxford, UK Thomas Kickler, MD: Johns Hopkins University Marlies Ledford-Kraemer, MBA, MT(ASCP)SH: CLOT-ED, Inc. Kandice Kottke-Marchant, MD, Ph. D: Cleveland Clinic Alvin Schmaier, MD: Case Western Reserve University Melanie Mc. Cabe 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 De. Biase: Sienco, Inc. Richard Marlar, Ph. D: Oklahoma City VA Medical Center Margaret Rand, Ph. D: 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 H 58 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 H 58 -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 De. Biase

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 -25 o. C), hand carried (no pneumatic tubes), and not be exposed to vibration, shaking or agitation PRP sample preparation for light transmission aggregometry (LTA) – – Centrifuge at 170 g 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 m. M 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 m. M 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 2 nd 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% 3% 4% 14% 4% 8% 5% 7% 6% 37%

Type of Aggregometry n=121 responses 59% (n=145/245) collected samples into 3. 2% sodium citrate (n=98) or buffered sodium citrate (n=47)

Pre-analytical Variables Patients are required to…… n=26/202 n=25/206 n=53/201 n=38/162

Needles Used For Sample Collection (n=179) n=2 n=49 n=101 n=27 • 32% (n=79/245) used a butterfly needle

Study a normal control in parallel with the patient

PRP Count Adjustment n=173 responses 149 adjust with PRP Value of Adjusted Counts n=119 responses

Aggregation and Secretion Studies Performed Together? n=153 responses n=24 n=29 n=100

Maximal Time Allowed for Completing LTA n=140 responses

Common LTA Agonists at least one concentration n=149 responses

Times for Monitoring LTA Responses Agonist (n) ADP Epinephrine Collagen Range (minutes) 3 – 10 3 - 10 Most common 5 minutes TRAP 3 - 10 PAF U 46619 Arachidonic Acid Gamma-thrombin Ristocetin 3 – 10 3 - 10 3 – 10 5 minutes (n=6) 10 minutes 5 minutes

Statistical Approach for Determining LTA RI

Parameters Evaluated for LTA

Parameters Included in Report