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

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 β2Gp1 (iii) Paraproteins, Eg IgA myeloma. Interfere with fibrin polymerisation

Kershaw G. ASTH 2014

• 1. Types of 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) Direct Oral Anti-Coagulants: Eg rivaroxaban (Xa)

argatroban (IIa); dabigatran (IIa); apixaban (Xa); edoxaban (Xa)

Kershaw G. ASTH 2014

…inhibitors

• A. Hereditary
• 2. Types of factor deficiency

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

• B. Acquired

….types of factor deficiency

• 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

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cross-linked stable clot

Kershaw G. ASTH 2014

Heparin/ATIII

LA

Rivaroxaban Dabigatran

FXIII

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

100% 50% + = 0% Patient Pool Nor 1:1 mix Example 1 Patient with single factor deficiency

APTT 45 secs 30 secs 32 secs (25-37)

~50% Mix APTT close to pooled normal = correction

Mix test as first investigation of a prolonged APTT

Kershaw G. ASTH 2014

Patient Pool Nor 1:1 mix

Example 2 Patient with inhibitor, eg LA or factor.

APTT 45 secs 30 secs 42 secs (25-37) ~50% inhibitor strength Mix APTT remains abnormal Y Y Y Y Y Y Y Y

+ =

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Mixing test interpretation for APTT

Immediate mixes: APTT 1:1 mix minus APTT Pooled normal

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Kershaw and Orellana; Semin Thromb Hemost 2013;39:283-290

Factor deficiency Inhibitors Non-correction Overlap Correction

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)

20 40 60 80 100 20 40 60 80 100 120

Minutes incubation at 37C

APTT

96

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!

20 40 60 80 100 20 40 60 80 100 120

Minutes incubation at 37C

APTT

Kershaw G. ASTH 2014

3 a 3 asp spects cts of

• f cl

clot

• tting

ting fa fact ctor

• r levels

ls

Level below which bleeding occurs Differs with factor

Level below which PT/APTT becomes prolonged 25-60%

Lower limit of reference range 50-70%

Thromb Haemost 2001; 85: 560 2001

Kershaw G. ASTH 2014

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

Classification of haemophilia A and haemophilia B

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.

50ul test plasma pre-diluted 1/10 + 50ul factor deficient plasma + 50ul APTT reagent

• incubate at 37°C 3-5 min

+ 100ul 0.025M CaCl2 Record time to clot formation. Read factor level from calibration curve.

Single cuvette set-up:

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One stage FVIII 6-100% calibration curve

1/10 1/20 1/40 1/80 1/160 1/8

Std dil’n

25 30 35 40 45 50 20 30 40 50 60 FS

Example of FVIII Sensitivity Curve

• the APTT upper limit (36sec) corresponds to ~45% FVIII:C

FVIII:C (prepared from 100%FVIII plasma

diluted in FVIII-deficient plasma) APTT Upper normal=36s

% factor (log scale) Factor assay calibration curve

• non-linearity effect of LA

Clotting Time (log scale)

6.3 12.5 25 50 100

. . . . .

95 86 75 64 56 Sample with inhibitor, eg LA Sample with no inhib

. .

Calibration curve

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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

1/10 1/20 1/40 1/80 1/160

Std dil’n

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FIX ‘Low’ calibration curve

• 1. Test plasma is diluted

1/40 in Tris-BSA buffer = source of FVIII

• 2. Remaining components
• f FIIa, FIXa, PL, Ca++ FX

and chromogenic substrate are sourced from kit.

FVIII IIa FVIIIa + FIXa + Phospholipid + Ca++ FX FXa

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1st stage: Generation of FXa

Chromogenic FVIII assay

FXa Chromogenic Peptide + pNA substrate (ΔOD 405nm over time)

(colourless) (yellow)

2nd stage : Measure how much Xa is produced Test components:

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Chromogenic FVIII 0-100% calibration curve

FVIII levels vary according to incubation time in patients with the discrepant phenotype in mild HA

Rodgers at al. Int. Jnl. Lab. Hem. 2009, 31, 180–188

1-stage clotting 2-stage clotting 2-st Chromogenic

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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

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CID et al

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Ratio of 1-stage clotting to Chromogenic FVIII assay

• 307 patients from 173 families:
• 2.8% families high ratio
• 9.8% families low ratio
• prevalence depends on selection
• f studied population

Results differed from other studies

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]

Case Study 1

2013/1

%

• 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]

Case Study 1

2013/1

Case Study 2

Male, 4 days old Mother is Haemophilia A carrier ? Haemophilia A

%

• 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]

Case Study 2

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.

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28 9 15 2.7 10 8.4 10 13 40 5 12 40 19 34 30 12 12 10.5 14.4 1-st clotting

2-stage chromogenic

19.6 2 12.9 3.9 8.4 3.7 9.2 4.4 45.9 4.6 5 53.3 15.9 23.9 24.1 13.1 4.1 5.2 13.2

Discrepant FVIII levels, % in mild HA

Note: There is no universally agreed definition of assay discrepancy.

Kershaw G. ASTH 2014

Factor inhibitors

Kershaw G. ASTH 2014

Basic laboratory characteristics of factor inhibitors

FVIII inhibitor Slow R n-c N N N FIX inhibitor Fast R n-c N N N FV inhibitor Fast R n-c R n-c N N Action APTT APTT PT PT TT Echis mix mix R, raised N, normal n-c, non-correction

Fast-acting vs slow-acting inhibitors

20 40 60 80 100 120 5 15 30 60 120 180 FVIII inhibitor ~1.5 BU/mL FIX inhibitor ~1 BU/mL FIX inhibitor ~4 BU/mL

% residual factor Incubation time (min) 1:1 mixes of test plasma : pooled normal incubated at 37°C

Kershaw G. ASTH 2014

• Type 1 inhibitors develop in

patients with congenital haemophilia A and are generally allo-antibodies that show complete neutralization of FVIII activity.

• Acquired inhibitors to FVIII show

type 2 kinetics, with a rapid neutralization phase, followed by an equilibrium in which residual FVIII activity can be detected in vitro

Kinetics of type 1 and type 2 inhibitors against factor VIII.

Ma, A. D. et al. Hematology 2006;2006:432-437

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Verbruggen, Seminars in Thrombosis and Hemostasis 2009, Volume 35, Number 8

The influence of incubation time on complex formation of factor VIII with inhibitor. Inhibitor activity is shown in Nijmegen-Bethesda units (NBU)/mL.

20 40 60 80 100 120 140 160 180 1 2 3 4 5 6

FVIII inhibitor APTT

BU/mL & seconds The APTT and FVIII inhibitor strength in acquired Haemophilia A Sample

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FVIII: levels at presentation – Acquired haemophilia

FVIII level at diagnostic (IU/dL)

• No. of patients reported in 2-y study period

for whom characteristic is known % Severe; 1 or less 46 29.87 Moderate; more than 1, less than 5 56 36.36 Mild; 5 or more, less than 50 52 33.77 Inhibitor titer category at diagnosis (BU/mL)

• No. of patients reported in 2-y study period

for whom characteristic is known % 0-10 9 6.25 11-100 119 80.56 101-1000 16 11.11

Collins at al. Blood, 2007; Volume 109, Number 5

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Fatal bleeding episodes in patients with acquired haemophilia A Time of death after presentation (days) Site of fatal bleed FVIII at presentation IU/dL Inhibitor titer at presentation BU/mL

Gastrointestinal <1 Not stated 2 Gastrointestinal 4 2 4 Lung 4 5 14 Intracranial 4 15 17 Postoperative 9 8 19 Retroperitoneal 2.3 18 24 Intracranial 9 1.4 66 Internal 5 219 106 Intracranial 4 6 136 Gastrointestinal <1 109 148 Intracranial 2 14 Not stated Retroperitoneal 12 4 Not stated Intracranial, retroperitoneal, gastrointestinal 3 6.4 Collins at al. Blood, 2007; Volume 109, Number 5

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Inverse relationship between Factor VIII level and Factor VIII Inhibitor Titre- Case study: Acquired haemophilia A

55% FVIII 0.91 BU/mL FVIII inhibitor

81 years old male patient with acquired haemophilia A

Coagulation factors: heat inactivation as a function of time

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Test plasma

Neat

1 in 2 1 in 4 1 in 8 …..etc Diluent Pooled normal

incubate 2 hours @ 37°C FVIII assay Residual FVIII = (patient tube FVIII/control tube FVIII) x 100 Final titre calculated from tube with residual closest to 50% Test plasma dilutions Nijmegen FVIII deficient plasma Imidazole buffered pH 7.4 ‘Classic’ Imidazole buffer pH 7.4 Unbuffered Control tube

Bethesda assay for FVIII inhibitors

• 3. Incubation
• 1. Sample dilutions + Normal Pool
• 4. Run samples for FVIII
• 2. Mixing

Bethesda Assay test set-up

FVIII Inhibitor Titre

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 10 100

% Residual

50 70 30

Titre BU/mL

1 BU is the amount

• f antibody that

neutralises 50% of the FVIII in a normal pool

Kershaw G. ASTH 2014

Kershaw G. ASTH 2014

%FVIII Titre %FVIII Titre %FVIII Titre Residual BU/mL Residual BU/mL Residual BU/mL 25 2.00 42.5 1.23 60 0.74 25.5 1.97 43 1.22 60.5 0.72 26 1.94 43.5 1.20 61 0.71 26.5 1.92 44 1.18 61.5 0.70 27 1.89 44.5 1.17 62 0.69 27.5 1.86 45 1.15 62.5 0.68 28 1.84 45.5 1.14 63 0.67 28.5 1.81 46 1.12 63.5 0.66 29 1.79 46.5 1.10 64 0.64 29.5 1.76 47 1.09 64.5 0.63 30 1.74 47.5 1.07 65 0.62 30.5 1.71 48 1.06 65.5 0.61 31 1.69 48.5 1.04 66 0.60 31.5 1.67 49 1.03 66.5 0.59 32 1.64 49.5 1.01 67 0.58 32.5 1.62 50 1.00 67.5 0.57

Conversion table for residual FVIII into Bethesda units; Final titre = raw titre x dilution factor Titre = [2-log10%R]/0.301

50

Post-incubation: FVIII in control tube = 50% (A)

Case Study: Weak inhibitor Sample Dilution

%FVIII remaining (B)

% Residual = (B/A)*100 Titre BU/mL Neat 2 6 1/2 10 20 1/4 24 48

1.06 x 4 = 4.2

1/8 40 80

Simple and complex inhibitor patterns

Kershaw G. ASTH 2014

The Bethesda Assay has poor inter-laboratory reproducibility

• 1. Coefficients of variation are very high in external QAP

eg CVs are 20-60% where CV = (SD/mean)*100; eg: at 2 BU/mL and 40%CV, 95% CI is 0.4 - 3.6 BU/mL

• 2. The Nijmegen version is the ISTH method but in reality very few

labs are using it. Even then, the CV are slightly lower than the

• ther methods.

Kitchen at al. Seminars in Thrombosis and Hemostasis 2009, Volume 35, Number 8

UK National External Quality Assessment Scheme human factor VIII inhibitor results by Bethesda assay: sample coded Congenital-

• 2007. The number of centers recording each particular inhibitor

result on the same sample is shown.

Kershaw G. ASTH 2014

Kershaw G. ASTH 2014

Kershaw G. ASTH 2014

Possible reasons why the Nijmegen method has not been more widely adopted:

• Too few assay performed in many labs
• Cost of Factor VIII deficiency plasma
• Most laboratories are in within “consensus” in

EQA so no need to change – self-perpetuating

• Paucity of samples for method validation

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Staff Pat FFP Dade ImmD IL STA vens’n Staff Pat FFP Dade Helena 14 5 4 11 1 1 1 2

4 3 1 4 3

• 2. Type NP

Breakdown of methods, n= 49; RCPA survey results

Owr Imm FVIII 5 7 1

• 3. Control tube dil.

Owr Imm FVIII 6 4 1 Owr sal 4 1 Imm FVIII 3 1 Imm 2 FVIII FVIII Alb FVIII FVIII 4 2 1 4 3

Many other differences noted Eg,- reading graph vs formula

• lower limit of reporting range 0.0 to 2.0
• whether patient’s FVIII checked
• acceptable FVIII level in normal plasma
• pH of NP
• brand of deficient plasma in FVIII assays

7/13 pH7.4 NP

Original Bethesda

n=36

Nijmegen assay

n=13

• 1. Method

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Kershaw, Chen, Jayakodi, Dunkley. Thrombosis Research 2013; 132; 735–741

Kershaw G. ASTH 2014

Validation of 4% albumin as a diluent in Bethesda assays

Change in FVIII inhibitor titre over a 240 day period time in an individual undergoing inhibitor tolerisation therapy as measured by the Nijmegen Bethesda assay (NBA) and the modified Nijmegen assay.

G.W. Kershaw et al. / Thrombosis Research (2013) 132 735–741

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FVIII 4% Albumin 5 BU

$15.34 $0.12

50 BU

$26.84 $0.21

250 BU

$34.51 $0.26

G.W. Kershaw et al., Thrombosis Research 2013; 132:735–741

$ Cost comparison

Sample & control tube diluent type

Kershaw G. ASTH 2014

Summary of Key Points

• Screening and Mixing tests are useful if deciding whether

factor deficiency or inhibitor is present …with some limitations.

• Knowledge of different factor deficiency and inhibitor

behaviour in adjunct tests is very useful in investigating coagulopathies.

• Bleeding tendency can be factor and patient specific.
• Chromogenic FVIII assays detect some mild HA cases missed

by 1-stage assays, and help define discrepant phenotypes. Initial screening by Chr assay is important.

• High inter-laboratory variability exists for Bethesda assays
• The Bethesda assay for FVIII inhibitors performs best when

individual laboratories standardise test conditions

Kershaw G. ASTH 2014

Any questions ?

Kershaw G. ASTH 2014