PhenX Measures: Inhibitors Barbara Konkle and Diane Nugent 4.18 - - PowerPoint PPT Presentation

PhenX Measures: Inhibitors

Barbara Konkle and Diane Nugent

4.18

Inhibitors

• Assays
• Existing Inhibitor State
• Evolving Inhibitor State
• Resolving or Persistent Inhibitor State

Laboratory Measures of Inhibitor Impact

• Factor VIII activity in plasma

– One-stage assay – Chromogenic assay

• Quantitative measure of inhibitor activity

– Nijmegen modification of Bethesda assay

• Measure of response to factor infusion

– PK study to assess initial factor recovery and half-life

FVIII Activity

• Definition: A bioassay to determine factor VIII

activity in plasma.

• Purpose: Factor VIII (FVIII) activity measured

in plasma determines the severity of hemophilia A. Values that are less than anticipated may reflect the presence of a neutralizing antibody (inhibitor).

Plasma FVIII Activity

• One-stage FVIII activity assay in plasma1,2

– Validated and widely available in clinical labs – Results can vary by reagent/instrument used

• Particularly for modified recombinant FVIII products
• Does not correlate with bleeding risk in setting of emicizumab
• Chromogenic FVIII activity in plasma1,2

– Less widely available in clinical labs – Generally, more uniform measures across laboratories1 – Chromogenic assay using bovine reagents not influenced by emicizumab

1Kitchen, et al. Haemophilia 2016;22(Suppl 5):72-77. 2Peyvandi, Oldenburg and Friedman. J Thromb

• Haemost. 2016;14:248-61.

Questions

• Do we state that FVIII should be measured by

chromogenic assay centrally?

– Would limit use of data collected locally

• Do we state that either can be used but

caveats noted?

Pre-analytical variables in FVIII measurement

• Blood collected by direct venipuncture into 3.2%

NaCitrate tube and filled correctly1

– Should be second tube collected. If no other tube drawn, a discard tube should be obtained. Blood should fill tube to within 33% of fill line. (11% if aPTT measured)

• Sample should be kept at RT and platelet poor

plasma prepared within 4 hours of collection.2

– Centrifuge should be validated so that process results in < 10,000 platelets/microliter – Can be stored at -20oC for 2 weeks, and should be transfered to <-70oC for longer storage, including shipment.

1Lippi et al, Quality standards for sample collection in coagulation testing.

Semin Thromb Hemost 2012;38:565-75.

2Funk et al, Quality standards for sample processing, transportation, and

storage in hemostasis testing. Semin Thromb Hemost 2012;38:576-85.

Bethesda Assay for Inhibitor Quantification

• Described in 1975 to quantify FVIII inhibitory activity.1
• Modified “Nijmegen modification,” to provide the

same buffering in patient sample and control and eliminate low titer false positives.2

• Further modified and validated to include a step to

heat and thus inactivate factor so that assay can be performed in patients on regular factor infusions.3

• Low titer: < 5 B.U.; high titer: > 5 B.U.4

1Kasper CK, et al. Thromb Diath Haemorrh 1975; 34 (3) 869-872 2Verbruggen B, et al. The Nijmegen modification of the Bethesda assay for factor VIII:C inhibitors: improved

specificity and reliability. Thromb Haemost 1995;73:247-51

3Miller CH, et al. Validation of Nijmegen-Bethesda assay modifications to allow inhibitor measurement during

replacement therapy and facilitate inhibitor surveillance. J Thromb Haemost 2012;10:1055-61.

4Blanchette VS, Key NS, Ljung LR, Manco-Johnson MJ, ven den Berg HM, Srivastava A. For the Subcommittee on

factor VIII, factor IX and rare coagulation disorders: Definitions in hemophilia: communication from the SSC of the

• ISTH. J Thromb Haemost 2014; 12: 1935-1939

PK Study to Assess Response to Factor Infusion

• Definition: A series of plasma factor VIII (FVIII) activity

determinations in blood samples obtained immediately prior to, and at time points after, infusion of FVIII concentrate.

• Purpose: A pharmacokinetic (PK) study is used to determine

the initial recovery and half-life of infused FVIII concentrate. International ITI study used before infusion and 15-30 minutes, and 1, 2, 4, 6, 24, and 48 hours after infusion.

Existing Inhibitor State

• Inhibitor Diagnosis

– Clinical: Lack of response (cessation of bleeding) to factor infusion for treatment of bleeding – Laboratory: Less than expected FVIII level and/or positive inhibitor titer in routine surveillance.

Evolving Inhibitor State

• Change in inhibitor titer over time

– Without immune tolerance induction – With immune tolerance induction

• Change in clinical response to factor infusion
• Change in factor VIII activity after factor

infusion

Resolving or Persistent Inhibitor State

• Inhibitor Resolution

– For patients receiving immune tolerance therapy for eradication of a FVIII inhibitor, success is defined as a negative FVIII inhibitor titer (Bethesda assay) and a normal recovery and half-life of infused FVIII concentrate. – Standard: Recovery ≥ 66% of expected and FVIII half-life ≥ 6 hr1 or 7 hr2

• Persistent Inhibitor

– Decrease response to FVIII concentrate infusion with or without a persistently positive inhibitor titer.

1Hay, DiMichele et al. Blood 2012;119:1335-44. 2Blanchette VS, et al. J Thromb Haemost 2014; 12: 1935-39.