Microangiophathic haemolytic anaemia (MAHA) & thrombcytopenic purpura (TTP) (BSH 2012)

Intro

Incidence of acute idiopathic TTP:  6 per million per year in UK

Untreated mortality:                         >90%

 

TTP Pathophysiology

 

Congenital deficiency of ADAMTS13, or acquired autoantibodies against ADAMTS13

 

ADAMTS13 – a metalloproteinase responsible for cleaving ultra large VWF multimers (ULVWF)

 

In the absence of ADAMTS13, ULWVF causes spontaneous platelet aggregation under conditions of high shear stress, e.g. microvasculature, brain, heart, kidneys.

Thrombocytopenia results from consumption within platelet-rich thrombi

Anaemia results from mechanical fragmentation of red cells through partly occluded vessels.

 

TTP Clinical Features

 

Classic Pentad – Fever, Thrombocytopenia, Renal Failure, Fluctuating neurology & MAHA

 

But:

33% No Neurological Signs

Fever and renal impairment may not be prominent

 

TTP Ix

 

FBC                             – median plt count 10-30 & Hb 80-100 at diagnosis

Film                             – thrombocytopenia, schistocytes

Haemolysis markers – haptoglobins, Retic, LDH, Normal DAT

Coag screen               – normal

Trop T                        – raised in 50%, poor prognostic sign

 

ADAMTS13 Assays

  • <5% +/- presence of an inhibitor confirms the diagnosis

  • TTP vs HUS – A level of <5% has 90% specificity for TTP

  • Activity, Antigen and Autoantibody assays available.

  • Activity assays based on the failure of patient plasma to degrade VWF multimers

 

Other Ix of MAHA/TMA

 

Pregnancy Test

Infection         – HIV, Hep B, Hep C

AI                    – TFT, Autoantibody screen

Cardiac           – ECG, Echo

Neuro             – CT/MRI Brain

Malignancy    – CT CAP, tumour markers

 

Differential Diagnosis of MAHA/TMA

 

Acquired TTP

Congenital TTP

  • 100 patients worldwide. Presents at any age. Often asymptomatic until a precipitating event sends a chronically low ADAMTS13 even lower.

Pregnancy

  • HELLP, Eclampsia, AFLP

  • Diagnostically difficult. Pregnancy may be precipitant for 5-25% of TTP cases

Haemolytic Uraemic Syndrome (HUS)

  • E. coli shiga toxins

Atypical HUS (aHUS)

  • Excess activation of alternate complement pathway

  • May be genetic, acquired autoantibody or idiopathic

Drugs

  • 15% of MAHA cases

  • TTP - Quinine, tacrolimus, simvastatin, interferon, OCP, trimethoprim

  • HUS – Gemcitabine, bleomycin, mitomycin-C

Malignant Hypertension

Malignancy

  • Adenocarcinoma especially. Early or late stage disease. ADAMTS13 not low.

Post-HSCT

  • Lack of ADAMTS13 deficiency, poor response to PLEX. Anecdotal use of defibrotide

Infection

  • CMV, Adenovirus, Herpes Simplex, Hep B, Hep C, Meningococcus, Fungal

  • HIV – may be presenting feature. ADAMTS13 <5% associated with a relatively higher CD4 count

Autoimmune

  • Lupus nephritis, Scleroderma, Evans Syndrome, Vasculitis

Catastrophic Antiphospholipid Syndrome (CAPS)

 

Treatment of Acute TTP

 

1. Start Plasma Exchange (PLEX) within 4 hours of diagnosis

Removes antibody and ULMW VWF Multimers

Replaces ADAMTS13

  • 1.5x plasma volume exchange daily for 3 days, then re-assess

  • Continue PLEX until plt count >150 (Complete remission) & for 2 more days beyond

  • Use SD-FFP to reduce risk of TTI & adverse immune responses.

  • (Note: MB-FFP associated with longer hospital stay and greater number of exchanges)

 

2. Start steroids

Stop production of antibody

  • 1g IV Methylprednisolone or 1mg/kg PO Prednisolone

 

3. Supportive Care

PPI

Folic Acid

LMWH + Aspirin once plt count >50 (SD-FFP deficient in protein S)

Red cell transfusion if required

Consider Hep B vaccination

 

Treatment of Specific Scenarios

 

Congenital TTP

  • Consider long-term prophylaxis – SD-FFP transfusion every 10-20 days

  • Supplement during pregnancy

  • Alternative: ‘8Y’  - a intermediate purity FVIII concentrate containing ADAMTS13

 

Relapse

  • Defined as an episode of acute TTP occurring >30 days after remission

  • Occurs in 20-50% of cases

  • Can be averted by ADAMTS13 monitoring and pre-emptive rituximab

 

Other therapies

  • 1st or 2nd line: Rituximab 375mg/m2 weekly for 4 doses

  • 2nd line: Ciclosporin, splenectomy

 

Further Details of Plasma Exchange

 

£3,000 per exchange (including cost of SD-FFP)

Two methods of separation - Centrifugal or Filtration

Removes ultra large vWF, replaces ADAMTS13 and removes inhibitor (e.g. antibody)

FFP contains around 1 unit ADAMTS13 per 1ml/kg. So 15ml/kg --> Raise ADAMTS13 by up to 15% (however rapidly consumed or deactivated by antibody)

Replace 1-1.5x plasma volume & takes 110-150 minutes per exchange

Flow rate around 120ml/min through machine

Centrifuge at 2400 rpm

ACD-A anticoagulant

Perform daily until platelet count >150 on 3 consecutive days, then wean off.

 

ADAMTS13 Indirect Activity ELISA

Technozym is one supplier. Takes 4-6 hours.

A microtitre plate is coated with anti-GST, subsequently bound to VWF73.

A source of ADAMTS-13 is then added (Calibrator, Control or Patient).

Cleavage of the substrate allows HRP antibody to bind to the remaining fragment --> colour change

adamts.png

 

ADAMTS-13 Inhibitor ELISA

 

To distinguish acquired from congenital TTP

Technozym is one supplier

A microtire plate is coated with recombinant ADAMTS-13.

When incubated with a source of ADAMTS-13 inhibitor (Claibrator, Control, Patient), the antibody will bind to ADAMTS-13.

Addition of an HRP antibody will produce a colour change

(Simple alternative: 50:50 mix with normal plasma)

adamst 2.png

 

Other ADAMTS-13 Assays

 

Antigen

  • may be normal in TTP, uninformative without activity assay

 

Direct Activity

  • SDS Gel Electrophoresis – incubate vWF with plasma and measure the drop in multimer size compared to diluted normal plasmas. Complicated and time consuming.

  • SDS PAGE & Western blotting – similar to above

  • FRET Assays – fluorescent resonance energy transfer

 

Anti-ADAMTS13 Autoantibodies

  • 50/50 mix with normal plasma