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
ISTH Advanced Training CourseDubai, UAE
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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ISTH Advanced Training CourseDubai, UAE
Which agonists?
Dawood BB, Lowe GC, Lordkipanidzé M, et al Blood 2012; 120 (25) 5041-9
ISTH Advanced Training CourseDubai, UAE
Defining reference ranges
Lowe GC, Lordkipanidzé M and Watson SP, Abstract presented at 14th UK Platelet Group Meeting, 2013- 4 -
ISTH Advanced Training CourseDubai, UAE
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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ISTH Advanced Training CourseDubai, UAE
PAR-1 peptide 30µM
Collagen 1µg/ml
Lowe GC, Sánchez Guiu I et al Thrombosis and Haemostasis 2013; 109(4):766-8- 6 -
ISTH Advanced Training CourseDubai, UAE
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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ISTH Advanced Training CourseDubai, UAE
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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ISTH Advanced Training CourseDubai, UAE
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ISTH Advanced Training CourseDubai, UAE
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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ISTH Advanced Training CourseDubai, UAE
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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ISTH Advanced Training CourseDubai, UAE
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ISTH Advanced Training CourseDubai, UAE
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 -
ISTH Advanced Training CourseDubai, UAE
Watson SP, Lowe GC, Lordkipanidzé M and Morgan NV, J Thromb Haemost 2013 11, (Suppl s1) 351-63
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ISTH Advanced Training CourseDubai, UAE
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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ISTH Advanced Training CourseDubai, UAE
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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ISTH Advanced Training CourseDubai, UAE
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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ISTH Advanced Training CourseDubai, UAE
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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ISTH Advanced Training CourseDubai, UAE
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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ISTH Advanced Training CourseDubai, UAE
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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ISTH Advanced Training CourseDubai, UAE
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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ISTH Advanced Training CourseDubai, UAE
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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ISTH Advanced Training CourseDubai, UAE
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 -
ISTH Advanced Training CourseDubai, UAE
• 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 -
ISTH Advanced Training CourseDubai, UAE
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 -
ISTH Advanced Training CourseDubai, UAE
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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ISTH Advanced Training CourseDubai, UAE
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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ISTH Advanced Training CourseDubai, UAE
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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ISTH Advanced Training CourseDubai, UAE
Thank you for listeningQuestions / Discussion
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ISTH Advanced Training CourseDubai, UAE
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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