case session: clinical investigation of patients with suspected ......lowe gc, lordkipanidzé m and...

ISTH Advanced Training CourseDubai, UAE

ISTH Advanced Training Course

Case session: Clinical investigation of patients with suspected platelet disorders

Advanced Training Course in Platelet Based Bleeding Disorders6th September 2016


Disclosures for In compliance with COI policy, ISTH requires the following disclosures to the session audience:

Research Support/P.I. No relevant conflicts of interest to declare

Employee No relevant conflicts of interest to declare

Consultant No relevant conflicts of interest to declare

Major Stockholder No relevant conflicts of interest to declare

Speakers Bureau Bayer plc

Honoraria Bayer plc

Scientific Advisory Board No relevant conflicts of interest to declare

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Which agonists?

Dawood BB, Lowe GC, Lordkipanidzé M, et al Blood 2012; 120 (25) 5041-9


Defining reference ranges

Lowe GC, Lordkipanidzé M and Watson SP, Abstract presented at 14th UK Platelet Group Meeting, 2013- 4 -


Case 1 77 year old Caucasian female

Pale skin and hair throughout life

Reduced visual acuity and nystagmus

Bleeding history ( ISTH BAT score 9) Spontaneous epistaxis requiring medical attention Prolonged bleeding from wounds requiring surgical

haemostasis Menorrhagia with hysterectomy aged 37 Bleeding post surgery requiring blood transfusion Multiple episodes of per rectal bleeding – right

hemicolectomy being considered

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PAR-1 peptide 30µM

Collagen 1µg/ml

Lowe GC, Sánchez Guiu I et al Thrombosis and Haemostasis 2013; 109(4):766-8- 6 -


Diagnosis: Hermansky-Pudlak syndrome Homozygous nonsense mutation in exon 4 of DTNBP1 –

HPS type 7 Identified by autozygosity mapping using microsatellite

markers

Learning points Patients can present with inherited conditions late in life Identification of underlying disorder may prevent

unnecessary medical interventions and surgical procedures

Aggregation alone may miss secretion defects Family history and family tree are crucial, but can be

difficult to broach in patients especially in first consult (consanguinuity is common in the West Midlands population, but varies significantly throughout regions in UK)

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Case 2 8 year old girl with easy bruising Local testing showed reduced maximal amplitude of

aggregation for all agonists. PFA closure times normal

Two siblings who bruise easily, and one with frequent epistaxis

Mother aged 39: Easy bruising with some spontaneous bruising.

Prolonged bleeding from minor wounds. No excessive bleeding with tonsillectomy. Received platelet transfusion prior to laparoscopy as an adult. Post partum haemorrhage requiring blood product (including platelet) transfusion after first of three deliveries

Maternal grandfather diagnosed with platelet storage pool disorder in Finland

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Right shifted collagen dose response curve and reduced response to intermediate dose PAR-1 peptide

Whole exome sequencing (of all family members samples) identified mutation in the ADP P2Y12 receptor DRY motif predicted to be function disrupting – R122H

Learning points: Importance of recruiting family members Ability to undertake platelet function testing in

children by rationalising agonist panel Bleeding symptoms in children may be regarded

as minimal due to insufficient challenges / exposures

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Case 3 64 year old Indian female

ISTH BAT score 13 Menorrhagia necessitating hysterectomy Excessive post-operative / post dental surgery bleeding Prolonged bleeding from minor cuts Easy bruising

Daughter with menorrhagia and sister with easy bruising – both unavailable for testing

Consistent absence in response to AA and reduced response with reversibility to U46619 (tested on multiple occasions)

Direct sequencing of TXBA2R – novel heterozygous missense mutation (N42S) in highly conserved region – predicted to be damaging / deleterious

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Cell expression studies revealed reduced ligand binding to TP receptor

Mutation associated with reduced cell surface expression intracellular retention of receptor

Learning points: Importance of patient recall Use of phenotyping to guide directed sequencing

when suspicion is strong / family members unavailable

Use of functional studies in confirming effect of mutation

Nisar SP et al Thrombosis and Haemostasis 2014 111(5):923-32 - 13 -


Watson SP, Lowe GC, Lordkipanidzé M and Morgan NV, J Thromb Haemost 2013 11, (Suppl s1) 351-63

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Case 4 29 year old Caucasian female

Long standing thrombocytopenia with platelet count around 30-40x109/l

Referred for pre-pregnancy counselling

Bleeding history: Epistaxis requiring cautery Easy bruising and prolonged bleeding from cuts requiring

surgical haemostasis Bled excessively after dental extraction and possibly after eye

surgery Menorrhagia Gum bleeding on teeth brushing despite good dental hygiene

Registered partially sighted (congenital cataracts, strabismus, detached retina)

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Case 4 Tinnitus and hearing loss

Poor steroid response

Declined splenectomy

Mother and two maternal half brothers thrombocytopenic

No neutrophil inclusions on blood film

Cyrus C. Hsia, AnargyrosXenocostas Blood 2012 119:32

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Whole exome sequencing revealed a MYH9 mutation (p.R1165C). This was confirmed by Sanger sequencing. Previous reports have shown this to be associated with renal impairment, deafness and cataracts.

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Learning points: Importance of querying congenital

thrombocytopenia when reduced platelet count is long standing, non responsive to immunosuppresion and present in many family members

Not all MYH9 mutations give neutrophilinclusions

Importance of being aware of syndromalassociations with low platelet count

Assessing function in thrombocytopenia is difficult and lumiaggregometry can be unreliable

Role of identification mutation in follow up planning and informing decision re pregnancy and its management

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Case 5 Approached by clinical geneticist

Asked for preconceptual advice: 24 year old lady Baby boy born by Caesarean section at 39 weeks, died a few hours after

birth Extensive haemorrhage and low platelet count Parents unrelated Bone marrow showed reduced numbers of megakaryocytes but no

suggestion of CAMT on initial genetic testing

Asked for approval to use post mortem samples in GAPP study for genetic analysis

Baby heterozygous for RUNX1 mutation - c.586 A>G:p.T196A.This lies within a region of the gene previously associated with thrombocytopenia

Mother wild type, father not available for testing

May explain bleeding and thrombocytopenia, but may not explain early death

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Case 5 Possible inheritance patterns: Inherited from father De novo mutation in utero Ovarian mosaicism

FBC in older children

Haematological involvement in future pregnancies

Possibility of further recruitment to 100000 genomes project for further information

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Learning points: Role of communication with bereaved parent and

ethics committee Caution in attributing all causality to identified

mutation Study information significantly added to

discoveries made within NHS investigation pathway

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Additional assays Demand for high throughput, standardised

platelet function tests which are not operator dependent and time consuming

Ideally could be done in whole blood / at the bedside

Potential applicability in children / post massive haemorrhage or trauma

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Whole blood flow cytometry• Remote testing of P-selectin and CD63 expression (alpha and dense granule markers)

• Use of patented fixative – stabilises for up to 9 days (Platelet Solutions, Nottingham)

Dovlatova N, Lordkipanidzé M, Lowe GC et al J Thromb Haemost. 2014 doi: 10.1111/jth.12555- 23 -


• Assessed in 48 healthy volunteers and 54 patients

• Overall diagnosis matched LTA in 83% of cases (kappa=0.667, p=0.02)

• Lacks kinetic information but may be helpful initial screening test to stratify who should have LTA

Whole blood flow cytometry

Dovlatova N, Lordkipanidzé M, Lowe GC et al J Thromb Haemost. 2014 doi: 10.1111/jth.12555- 24 -


Optimul assay: 96-well plate aggregometry

Plate coated with:◦ AA, ADP, Coll, Epi, TRAP,

U46619 and Ristocetin

Slide courtesy of Marie Lordkipanidzé, technique from Prof Tim Warner- 25 -


Comparison with lumiaggregometry

n=15 Median, IQRLordkipanidzé M, Lowe GC, Kirkby NS et al Blood 2014 Feb 20;123(8):e11-22

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Utility in inherited bleeding disorders

Lordkipanidzé M, Lowe GC, Kirkby NS et al Blood 2014 Feb 20;123(8):e11-22

• n = 65 patients– Platelet defect was detected by

LTA in 33/65 (51%) – Platelet defect was detected by

Optimul in 39/65 (60%)

• Good agreement between assays– Concordant: 82%– κ = 0.630, p <0.0001

OptimulLTA

Normal Defect

Normal 23 9Defect 3 30

• Predictive values– Sensitivity:

– 91% (95%CI 76-98)

– Specificity: – 72% (95%CI 53-86)

– Positive predictive value: – 77% (95%CI 61-89)

– Negative predictive value: – 88% (95%CI 70-97)

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Conclusions Clinical information is crucial in predicting

likely abnormalities and guiding testing

Appropriate phenotyping informs genetic testing

Where possible cell line / expression studies can confirm functional effects of mutations

Importance of multidisciplinary involvement in platelet clinical research

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Thank you for listeningQuestions / Discussion

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Acknowledgements Birmingham Platelet Group◦ Marie Lordkipanidzé, Natasha Dovlatova, Neil

Morgan, Ban Dawood, Danai Bem, Steve Watson, Paul Harrison, Gayle Halford

GAPP consortium◦ Andrew Mumford, Martina Daly, Stuart Mundell, Paul

Gissen, Sue Fox Birmingham and the Black Country CLRN West Midlands Ethics Committee Non-malignant haematology NIHR subgroup All research site Principal Investigators and Research

and Development administrative staff Haemostasis team, University Hospital Birmingham

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case session: clinical investigation of patients with suspected ......lowe gc, lordkipanidzé m and...

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