Laboratory diagnosis of haemophilia and inhibitors. Geoffrey - PowerPoint PPT Presentation

Laboratory diagnosis of haemophilia and inhibitors. Geoffrey Kershaw Royal Prince Alfred Hospital NSW Kershaw G. ASTH 2014 ASTH, Melbourne 22.03.2013

Presentation Outline 1. Classification of inhibitors and deficiencies 2. Screening and mixing tests 3. Factor assays - FVIII 4. Bethesda assays for factor inhibitors 5. External QAP 6. Summary Kershaw G. ASTH 2014

1. Types of inhibitors A. IMMUNOGLOBULINS (i) Factor inhibitors: removal of specific clotting factors by binding and/or neutralisation Eg. FVIII inhibitor - allo-antibodies (congenital HA) - auto-antibodies (acquired HA) (ii) Lupus anticoagulants: Ab’s bind phospholipids slowing clotting factor activity, prolong clotting times. eg β 2Gp1 (iii) Paraproteins, Eg IgA myeloma. Interfere with fibrin polymerisation Kershaw G. ASTH 2014

…inhibitors B. THERAPEUTIC AGENTS - designed to inhibit pro-coagulant activity in vivo and which have corresponding in vitro effects on clotting times. (i) Heparin, LMWH, Fondaparinux: act through antithrombin to bind and neutralise various activated clotting factors (ii) Hirudins eg lepirudin, bivalirudin: directly inhibit thrombin (iii) D irect O ral A nti- C oagulants: Eg rivaroxaban (Xa) argatroban (IIa); dabigatran (IIa); apixaban (Xa); edoxaban (Xa) Kershaw G. ASTH 2014

2. Types of factor deficiency A. Hereditary Haemophilia A ~ 1/ 5,000 male births Haemophilia B ~ 1/30,000 male births Mild FXII deficiency is relatively common (20-40% FXII) Severe FXII deficiency sometimes seen. (<1% FXII) Deficiencies of other factors much rarer, perhaps 1/million for severe cases of FII, FV, FVII, FX deficiency Occasionally encounter heterozygotes of these. Kershaw G. ASTH 2014

….types of factor deficiency B. Acquired 1. Liver disease. Reduced synthesis Variable PT/APTT patterns of prolongation All factors reduced, but FVIII usually normal/high Prot C, Prot S and AT also reduced. 2. Vitamin K deficiency or Vitamin K antagonists Reduced functionally active form of FII,VII,IX & X, PC,PS Raised PT and DRVVT; normal or raised APTT. 3. Associated with other clinical conditions eg - FX deficiency due to absorption by amyloid - FXII and AT deficiency in nephrotic syndrome - DIC: consumption of multiple factors Kershaw G. ASTH 2014

LA Heparin/ATIII Rivaroxaban Dabigatran FXIII Kershaw G. ASTH 2014 cross-linked stable clot

Screening tests for inhibitors and factor deficiencies 1. PT 2. APTT 3. FIBRINOGEN 4. THROMBIN TIME 5. FBC/platelets film 6. MIX test immed. and prolonged incubation - required for FVIII inhibitors 7. Factor assays Specific tests for inhibitors Inhibitor Titre (Bethesda) assays for factor inhibitors Lupus anticoagulant assay Need to exclude anticoagulant drugs Kershaw G. ASTH 2014

Investigation of a prolonged clotting time MIX TEST Correction Non- correction =Factor deficiency =Inhibitor Q. Is it really that simple? A. Often it is, but not always Kershaw G. ASTH 2014

Mix test as first investigation of a prolonged APTT Example 1 Patient with single factor deficiency 100% 50% + = ~50% 0% Patient Pool Nor 1:1 mix APTT 45 secs 30 secs 32 secs (25-37) Mix APTT close to pooled normal = correction Kershaw G. ASTH 2014

Example 2 Patient with inhibitor, eg LA or factor. Y Y Y Y Y + = ~50% inhibitor strength Y Y Y Patient Pool Nor 1:1 mix APTT 45 secs 30 secs 42 secs (25-37) Mix APTT remains abnormal Kershaw G. ASTH 2014

Mixing test interpretation for APTT Kershaw and Orellana; Semin Thromb Hemost 2013;39:283-290 Factor deficiency Immediate mixes: Inhibitors APTT 1:1 mix minus Non-correction APTT Pooled normal Overlap Correction Kershaw G. ASTH 2014

Effect of FVIII inhibitors on APTT mixing tests Case 1: Patient KL severe HpA Titre = 59 BU/mL, Pool normal APTT = 29 sec Patient APTT = 96 sec Immediate 1:1 mix = 46 sec hour 37ºC mix = 76 sec (still rising) 100 96 80 APTT 60 40 20 0 20 40 60 80 100 120 Minutes incubation at 37C

Effect of FVIII inhibitors on APTT mixing tests Case 2: Patient G Titre = 32 BU/mL, Pool normal APTT = 29.5 sec Patient APTT = 85.9 sec Immediate mix = 65.8 sec The 1:1 mix APTT reached maximal value with 10min incubation! 100 80 APTT 60 40 20 0 0 20 40 60 80 100 120 Minutes incubation at 37C Kershaw G. ASTH 2014

3 a 3 asp spects cts of of cl clot otting ting fa fact ctor or levels ls Level below which Lower limit of bleeding occurs reference range 50-70% Differs with factor Level below which PT/APTT becomes prolonged 25-60%

Thromb Haemost 2001; 85: 560 2001 Kershaw G. ASTH 2014

Classification of haemophilia A and haemophilia B White at al. Thromb Haemost 2001; 85: 560 Factor Level Classification <0.01 IU/ml (<1% of normal) severe 0.01-0.05 IU/ml (1%-5% of normal) moderate >0.05-<0.40 IU/ml (>5%-<40% of normal) mild 1 IU/ml of factor VIIIC (100%) is international standard for Plasma Factor VIII:C according to WHO Kershaw G. ASTH 2014

APTT-based automated factor assay. Single cuvette set-up: 50ul test plasma pre-diluted 1/10 + 50ul factor deficient plasma + 50ul APTT reagent -incubate at 37 ° C 3-5 min + 100ul 0.025M CaCl 2 Record time to clot formation. Read factor level from calibration curve .

One stage FVIII 6-100% calibration curve Std dil’n 1/10 1/20 1/40 1/80 1/160 1/8 Kershaw G. ASTH 2014

Example of FVIII Sensitivity Curve -the APTT upper limit (36sec) corresponds to ~45% FVIII:C 50 45 40 APTT Upper normal=36s FS 35 30 25 20 30 40 50 60 FVIII:C (prepared from 100%FVIII plasma diluted in FVIII-deficient plasma)

Factor assay calibration curve -non-linearity effect of LA . 95 . Sample with inhibitor, eg LA Clotting 86 . . Time . (log scale) 75 . 64 . Calibration curve 56 Sample with no inhib 6.3 12.5 25 50 100 % factor (log scale)

One stage FVIII ‘Low’ calibration curve Bench top pre-dilution of calibrator 1 in 10 in FVIII deficient plasma allows better determination of very low factor levels Std dil’n 1/10 1/20 1/40 1/80 1/160 Kershaw G. ASTH 2014

FIX ‘Low’ calibration curve Kershaw G. ASTH 2014

Chromogenic 1 st stage: Generation of FXa FVIII assay FVIII Test components: IIa 1. Test plasma is diluted FVIIIa + FIXa + Phospholipid + Ca ++ 1/40 in Tris-BSA buffer = source of FVIII FX FXa 2. Remaining components of FIIa, FIXa, PL, Ca ++ FX and 2 nd stage : Measure how much Xa is produced chromogenic substrate FXa are sourced from kit . Chromogenic Peptide + pNA substrate ( Δ OD 405nm over time) (colourless) (yellow) Kershaw G. ASTH 2014

Chromogenic FVIII 0-100% calibration curve Kershaw G. ASTH 2014

FVIII levels vary according to incubation time in patients with the discrepant phenotype in mild HA 2-st Chromogenic 1-stage clotting 2-stage clotting Rodgers at al. Int. Jnl. Lab. Hem. 2009, 31, 180 – 188

20% of 163 patients with mild HA had 15% higher 1-stage FVIII levels 5% higher chromogenic levels Tendency of bleeding to reflect more the FVIII level measured chromogenically Kershaw G. ASTH 2014

CID et al Kershaw G. ASTH 2014

Ratio of 1-stage clotting to Chromogenic FVIII assay Results differed from other studies • 307 patients from 173 families: • 2.8% families high ratio • 9.8% families low ratio • prevalence depends on selection of studied population Kershaw G. ASTH 2014

Case Study 1 Male, 18 months -with prolonged bleeding with mouth injury Hb 107 g/L WCC 14.6 x10^9/L Platelets 244 x10^9/L MCV 80 fL Hct 0.30 L/L Ferritin 17 ug/mL [10-150] PT 13.5 sec [11-15] APTT 45.9 sec [23-34] Fibrinogen 2.2 g/L [1.5-6.0] 2013/1

Case Study 1 % Ref. Interval HA FVIII:C 1-stage assay 7.3 [70-220] mild FIX:C 72 [50-200] FVIII chromogenic 4.1 [70-220] moderate VWF:Ag 101 [55-200] VWF:Ac (rGp1b) 83.6 [50-185] VWF:CB 105 [45-180] 2013/1

Case Study 2 Male, 4 days old Mother is Haemophilia A carrier ? Haemophilia A

Case Study 2 % Ref. Interval HA FVIII:C 1-stage assay 37 [70-220] mild FVIII chromogenic 27 [70-220] mild FVIII ratio = 27/37 = 0.73 VWF:Ag 140 [55-200] VWF:Ac (rGp1b) 152 [50-185] VWF:CB 146 [45-180] 2013/2

Case Study 3 Male, 25 yrs Mild HA FVIII:C levels 8-10% Retested by chromogenic assay: 1-stage 9% mild Chromogenic 2% moderate 6 of 32 mild HA had discrepant phenotype when re-assessed by chromogenic assay. All were lower by Chromogenic assay.

1-st clotting 2-stage chromogenic 28 19.6 9 2 Discrepant 15 12.9 2.7 3.9 10 8.4 FVIII levels, % 8.4 3.7 10 9.2 13 4.4 in mild HA 40 45.9 5 4.6 12 5 40 53.3 19 15.9 Note: 34 23.9 There is no universally 30 24.1 agreed definition of 12 13.1 assay discrepancy. 12 4.1 10.5 5.2 14.4 13.2 Kershaw G. ASTH 2014

Factor inhibitors Kershaw G. ASTH 2014