• Needs in hemophilia for the accurate measurement of hemostasis in patients include:

a) Patient Diagnosis

- correct assignation of clinical severity
- choice of appropriate therapy (e.g. prophylaxis, gene therapy)
- ability to monitor changes in disease severity

b) Patient Treatment

- need to effectively monitor and control replacement therapy w/r/t

• bleeding prophylaxis
• complications of therapy (e.g. thrombotic potential with bypass therapy)
• design of minimum effective dosing protocols for cost effectiveness

c) Standardization of replacement product by manufacturing by more sophisticated analysis

• Suggested ways to meet these needs include:

a) development of assays that can be held to performance standards

- easily performed in a clinical laboratory
- more accurately reflective of in vivo biological clotting activity
- involve dissociation of clotting endpoint and thrombin generation

b) validation of these assays in

- hemophilic dog models
- clinical patient studies

• however clinical outcome efficacy scales need to be improved to allow for objective vs. subjective assessment

• Is there such a state as a patient hemostasis baseline?
• Is a patient hemostasis baseline required for accurate therapy?
• Is a patient hemostasis baseline predictive of clinical bleeding phenotype?
• Does a predictive clinical phenotype exist?

• status of experimentation
• advantages/disadvantages of the individual assay methodology

• The ideal assay for hemostasis measurement:

- good signal for clot formation
- physiologic, i.e. TF-dependent endpoint
- sensitive enough at the thresholds for both bleeding and hypercoagulability

• Principles of the assay

- clotting velocity is a critical endpoint
- TF is crucial for sensitivity
- system amplitude is affected by cellular components, especially platelets

• Clinical correlation studies

- heterogenicity in biochemically severe (< 1%) hemophilia patients established
- clear dose response curve established in recombinant factor VIII spiking experiments (normal measurements achieved at 0.05 u/ml factor concentration)

• Future studies need to establish

- individual patient baseline variability with serial sampling
- clot stability endpoints in dose response curve experiments

• Potential assay disadvantages

- samples need to be run fresh; therefore it would be difficult to include this assay in collaborative assay comparison studies

• Interim data (obtained in collaboration with the London Royal Free Hospital Haemophilia Center)
• Principles of the assay

- not TF-based assay
- thrombin generation in the assay modified by concentrations of IXa, Xa, VII and TFPI
- thrombin generation measured by:

• T max reaction kinetics (lag phase and initial rate):  (potentially the most sensitive measurement)
• AUC (potentially less sensitive)

• Clinical correlation studies

- factor VIII reconstitution of plasma samples shortens reaction time (T max) but does not alter TG peak, AUC

• Future studies need to establish

- whether this assay may be more useful as a concentrate assay than a patient plasma assay

• Potential assay advantages

- Technically simple

• few reagents required
• adaptable to semi-automated instrumentation or to manual technology
• potentially useful in the developing world

- Flexibility

• can be adapted to different purposes
• applicability in both hemophilia A/B

- Sensitivity

• can factor detect levels of < 1 u/dl

• Potential assay disadvantages

- labor intensity
- time consumption (20 – 30 min/assay)
- difficulty in automation
- decreased precision compared to chromogenic TGT

• Potential applications

- more precise biochemical phenotyping
- post infusion studies/characterization of hemostasis
- monitoring of gene therapy

• Main questions:  Is there a problem with the PTT-based clotting assay per se or simply in our interpretation of the assay?
• Principles of the assay

- measurement of changes in signal intensity; timing of events; and velocity acceleration of the clotting reaction
- all phases of clotting measured:

• pre-clot formation (a-b)
• clot formation (b-d)
• post-clot formation (d-e)

• Clinical correlation studies (Drs Shima and Yoshioka)

- among 5 severe HA plasmas:  different waveform patterns noted, with the highest min-2 derivative seen in the least severe bleeder
- unlike the clot endpoint APTT, correlation of this assay in the hemophilia patient with plasma concentrates to a lower limit of 0.2%
- factor reconstitution of depleted plasma:  correlations of waveform pattern with [FVIII] between 0.1 – 1%

• Future studies

- analytical parameters other than min-2 must now be examined for sensitivity/specificity
- further work must be done with hemophilia B plasmas
- effect of different APTT reagents on analytical correlations must be examined
- potential adoption of this method to a PT-based assay

• Based on 1) the observation that clinical phenotype variability is most commonly observed among these hemophilia patients classified as biochemically severe (< 1%) by current assays; 2) the opinion that the greatest need for phenotype prediction with respect to therapeutic intervention lies within this subgroup of patients and 3) the consequent assumption that this group of patients is a priority target group for clinical/assay correlation studies; the following bleeding phenotype definition was proposed for this population:

- "Severe" Severe:  > 3 FVIII infusions required for spontaneous bleeding during the first year of life and > 3 joint hemorrhages; prophylaxis started by the age of 3.

- "Mild" Severe:  £ 3 FVIII infusions for spontaneous bleeds during the first year of life and £ 3 joint bleeds by the age of 3, in the absence of prophylaxis.

• Discussion

- alternatively, should clinical criteria be developed in the absence of biochemical criteria (i.e. not tied into our current biochemical definitions of severity)?
- what defines a "spontaneous" vs. "traumatic" bleed?  (so defined in the proposal to rule out bleeding due to e.g. traumatic venipuncutre or circumcision which is more circumstantial that inherently defining of the phenotype)
- should there be an infusion requirement to define a significant bleed?  (so defined in the proposal to exclude mild unconsequential bleeding such as non-progressive hematomas)
- should "non-iatrogenic" be used instead of the word "spontaneous"?
- how can cultural differences in bleed recognition/treatment (e.g. developing vs. developed world) be incorporated into the above definition?

1. Collaborative assay studies should be designed/done, acknowledging that some assays (rTEG) may not be adaptable to multicenter studies.
2. Collaborative multicenter studies could potentially include all of the following assay systems which can utilize frozen plasma samples:

- clotting/chromogenic factor assays
- TGT
- waveform PTT
- computer modeling programs

3. Limited correlation of these assay data with the rTEG could be done
4. Samples of 1 ml of frozen plasma would suffice for (each?) assay
5. Although the potential for individual patient variability in "baseline hemostatic" status exists and has not yet been studied by any of these assays, the priority for any collaborative studies should be to establish diagnostic and clinically predictive hemostasis baselines
6. Hemostasis baselines are critical to the future study of therapeutic intervention studies, including minimum dosing studies
7. For hemostasis baseline studies

- hemophilia A is the priority disease given frequency, clinical phenotype variability and greater therapeutic challenges
- study subject age must be considered relative to the developmental coagulation physiology in infants and young children and potential puberty-induced changes in adolescents

8. For therapeutic (post-infusion) studies

- both plasma-derived and recombinant products should be studied
- target factor levels of between 50 – 100% should be initially correlated with the proposed alternative assays