Pearls and Pitfalls in Factor Inhibitor Testing

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1 Pearls and Pitfalls in Factor Inhibitor Testing Dorothy M. Adcock M.D. Esoterix Coagulation, Laboratory Corporation of America Holdings ISLH May 19, 2015

2 Outline Overview of coagulation factor inhibitors Review of the incubated mixing test Review of the Bethesda assay and Nijmegen modification Example cases Potential causes of false positive and false negative factor inhibitor assays Safeguards in testing

3 Coagulation Factor Inhibitors Polyclonal immunoglobulins that bind and neutralize the activity of a coagulation factor Not all antibodies are functional neutralizers Develop in patients with congenital factor deficiency in response to replacement therapy or as an autoimmune phenomenon Can be life-threatening and/or lead to significant morbidity False positive and negative results can lead to serious patient mismanagement Proficiency testing surveys* reveal 32% false positive and 5% false negative results; with no improvement over the past decade *ECAT, NEQAS, RCPA QAP

4 Assays to Detect and Measure Coagulation Factor Inhibitors 1959: Oxford Assay; considered unreliable Uses Cryoprecipitate as the FVIII source 1975: Bethesda assay; allowed an acceptable degree of standardization but considered rather non-specific * (many false positive results) Uses NPP as FVIII source Uses NPP + imidazole buffer as reference 1995: Nijmegen assay; decreases false-low positive results Uses buffered NPP Replaces imidizole with FVIII deficient plasma * Hemophilia 2014, 20 (Suppl.4), 94-98

5 Normal Plasma Mixing Test Factor inhibitor assays [Bethesda assay (BA)/Nijmegan (NBA] are based on a normal plasma mixing test Causes These are of an the abnormal very same mixing causes test of (lack a of correction false positive into BA/NBA the normal reference interval): Factor inhibitor* Lupus anticoagulant* EDTA plasma* Direct Oral Anticoagulants (DOACs)* Heparin *May demonstrate prolongation with incubation

6 Incubated Mixing Test 50% 50% Incubate each at 37 C for 60 to 120 minutes, then mix patient with NPP Patient NPP Patient + NPP Compare Incubated Mix to Control Separately incubated patient + NPP Mix (Control) Patient + NPP Incubated together (Incubated Mix)

7 Sample Plasma Mixing Study Table Results in Seconds FVIII Inhibitor Fast Slow FVIII Deficiency Lupus Anticoagulant FIX Inhibitor APTT Normal APTT 1:1 immediate APTT 1:1 Incubated NP Control APTT 1:1 Incubated NP Patient

8 Bethesda Assay (BA) Neat 1:2 1:4 1:8 Patient 37 C Perform Factor Activity 1:10 & 1:20 Patient + NPP Control Determine RA% = Patient Mix Control Buffer + NPP 37 C Perform Factor Activity RA must fall between 25% and 75% to call a result positive and to calculate a titer

9 Determining Bethesda Titer Corrected Percent Residual Factor Activity Bethesda Units Per ml Plasma Graphically or by using Excel Bethesda Unit (BU) = ( 2-log %RA)/ Correct for dilutions if needed! RA must fall between 25% and 75% RA <25%: make additional dilutions RA >75%: BA is negative and this equates to 0.4 BU Internationally agreed upon detection limit 0.6 BU* * Hemophilia 2014, 20 (Suppl.4), 94-98

10 Bethesda Assay Methodology with Nijmegen Modification Neat 1:2 1:4 1:8 Patient Patient + NPP Buffered NPP Control Factor Buffer DP + NPP + NPP Nijmegen modification uses buffered normal plasma in the patient mix and Factor DP (or 4% albumin)* rather than buffer in the control mix Increase in ph leads to inactivation of FVIII and this is further enhanced with reduced protein concentration Nijmegen modification increases specificity and assay reliability *Giles AR, et al. Thromb Haemost 1998;79:872-5

11 BA Calculation Sheet Example Patient Dependent Dilution FVIII Bethesda Assay Calculation Sheet Patient Tube Factor VIII Activity 1:10 1:20 Av Control Tube Factor Activity %RA Log Residual Activity BU BU X dilution factor Smith 1 Vmax Vmax ncalc 45 ncalc ncalc ncalc ncalc 1:2 Vmax Vmax ncalc 45 ncalc ncalc ncalc ncalc 1:5 5.2 Vmax : : : : :

12 Causes of False Positive BAs Perfect mimic a FVIII inhibitor EDTA Plasma Decrease in FVIII activity, lack of correction with 1:1 mix, prolongation with incubation and positive NBA Causes of Positive NBA but with Non- Parallelism Factor inhibitor in a BA other than the assay of involved factor Such as a FVIII inhibitor in a FIX or FXI NBA Lupus anticoagulant Direct oral anticoagulants Heparin

13 Specific Factor Inhibitor Present but is Other than the Factor Inhibitor Under Investigation A strong FVIII inhibitor: Can inhibit the FVIII in a FIX or FXI activity assay causing factor IX or XI to appear spuriously low Appears as a non-specific inhibitor as it can be diluted Can cause a false positive FIX or FXI BA Typical BA results, if run on all intrinsic factors: Bethesda Assay Bethesda Titer Factor VIIII 850 Factor IX 13 Factor XI 18

14 Example: FVIII Inhibitor in a FIX BA FIX Bethesda Assay Calculation Sheet Patient Dependent Dilution Patient Tube Factor IX Activity Control Factor Activity %RA Log Residual Activity BU BU X dilution Factor 1:10 1:20 Av Jones ncalc ncalc ncalc ncalc 1: ncalc ncalc ncalc ncalc 1: *** 53 ncalc ncalc ncalc ncalc 1: *** 53 ncalc ncalc ncalc ncalc 1: *** 53 ncalc ncalc ncalc ncalc 1: *** 53 ncalc ncalc ncalc ncalc ***If non-parallelism occurs, further investigation is needed.

15 Example: FVIII Inhibitor in a FIX BA Activities of Dependent Dilutions Performed at One Dilution Only Patient Dilution Patient Tube Factor IX Activity Factor IX Bethesda Assay Calculation Sheet Control Factor Activity %RA Log Residual Activity BU BU X dilution Factor Jones ncalc* ncalc ncalc 1: ncalc ncalc ncalc 1: : : : Potential for reporting false positive BA result

16 Safeguards in Performing Bethesda Assays Characterize the plasma sample by performing APTT, PT and thrombin time Rule out EDTA Rule out anticoagulant drug effect Properly perform and interpret incubated APTT mixing test and correlate this with results Perform the Nijmegen modification rather than the standard Bethesda assay Perform the dependent dilutions of the NBA at 2 dilutions and investigate non-parallelism

17 Thank you for your attention Dorothy M. Adcock