platelet esoteric testing: when and why...11 ©2020 mfmer | slide-21 ngs testing on both blood and...

12/3/2020

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©2020 MFMER | slide-1©2020 MFMER | slide-1

Platelet Esoteric Testing: When and Why

Dong Chen MD PhDSpecial Coagulation LaboratoryDivision Of Hematopathology

A Case-based Workshop: Clinical and Laboratory Aspects of Hemophilia and Thrombosis Dec 4th, [email protected]

©2020 Mayo Foundation for Medical Education and Research | slide-1

©2020 MFMER | slide-2

DISCLOSURES

Relevant Financial Relationship(s) • None

Off Label Usage• None

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LEARNING OBJECTIVE

1. Know the platelet laboratory testing algorithm

2. Understand the platelet esoteric testing

3. Use 2 cases to illustrate our experiences


Introduction1

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Diagnosis of Inherited Platelet Disorder

Clinical Assessment Standard Laboratory Testing Esoteric Laboratory Testing

• CBC and Morphology• Peripheral blood CBC

and platelet indices• Peripheral blood smear

• Platelet Functional Tests• Platelet aggregation • Platelet function analysis

(PFA-100) • Granule release

(serotonin or ATP release)

• Patient’s bleeding and other history

• Bleeding assessment• Other histories

• Cardiovascular• Bone marrow status• Malignancy• Neuromuscular• Vision and

pigmentations

• Family history• Bleeding history• Other histories

• Esoteric Testing• Flow cytometry• Transmission electron

microscopy• Genetic testing

Diagnosis


Platelet Surface Receptor Assessment by Flow Cytometry

TXA2, ADP receptors

Collagen receptor deficiency

Glanzmann thrombasthenia

Collagen receptor deficiency

Bernard-Souliersyndrome

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Quantification of Platelet Surface Glycoproteins by Flow Cytometry

CD41 expression level (%) = (MFI: Mean fluorescent intensity

Platelets

Red cells

n=112; age:18-76 years


Glanzmann Thrombasthenia

GPIIb GPIIIa

GPIXGPIB-α GPIa/IIa

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Platelet Transmission Electron Microscopy (PTEM)

• Whole Mount• Dense granules

• Thin Section• Alpha granules• Other platelet ultrastructure

• Buffy Coat• Inclusions in leukocytes


Platelet Transmission Electron Microscopy (Normal Donor)

DGAG

MT

Gly

DG

AG: Alpha granule

DG: Dense granule

Gly: Glycogen

MT: Mitochondria

Whole mount PTEM Thin-section PTEM

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Claire Lentaigne et al. Blood 2016;127:2814-2823

Molecular Testing of Inherited Platelet Disorders


TIER169

• Proven disease-associated gene (DIAGNOSTIC-GRADE)• 3 unrelated pedigrees with co-segregation data• Functional data (animal model and cell/protein studies)

TIER2• Evidence from 1 or 2 pedigrees with insufficient co-

segregation data & without functional studies

TIER3• Evidence for role in platelet disorders (e.g. Functional

studies or KO mice)

www.isth.org/page/GinTh_GeneLists

Quan Li and Kai Wang. InterVar: Clinical interpretation of genetic variants by ACMG-AMP 2015 guideline

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Case illustration2Case 1Bleeding and life-long thrombocytopenia

Case 2Bleeding and life-long thrombocytopenia


Case 1

A 51-year-old woman with life-long thrombocytopenia

Patient History

• She had severe epistaxis and was found to have thrombocytopenia

• Her platelet count has been 30,000-90,000/μL

• Her ISTH bleeding score is 9: menorrhagia, epistaxis requiring ENT intervention, significant bruising

• She had 2 vaginal deliveries, and both had excessive bleeding requiring platelet transfusions

• She had a bone marrow biopsy which was morphologically normal

• She had hysterectomy for a uterine mass and endometriosis. Her postoperative course (2 months) was complicated by bleeding, which required two surgical re-explorations and more than 10 units of platelet transfusion

6Age

49

51

20s

30s

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Family History

No family or personal history of immune deficiencies, bone or skeletal abnormalities, skin disease or warts, deafness, renal failure, idiopathic pulmonary fibrosis, or cirrhosis.


Standard Laboratory Testing

CBC CoagulationHemoglobin (g/dL) 12.5 INR 1.0Hematocrit (%) 37.4 APTT (sec) 33Platelet count (10^9/L) 20 ↓ Thrombin time (sec) 16MPV (fL) 11.8 ↑ VWF antigen (IU/dL) 150White blood cell (10^9/L) 4.6 Fibrinogen (mg/dL) 342

VWF activity (IU/dL) 138Platelet Laboratory Testing

PFA-100 Canceled* Platelet aggregation Canceled*

* Due to low platelet count.

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Peripheral blood smear


Platelet Glycoprotein Assessment by Flow Cytometry

GPIIb GPIIIa

GPIXGPIB-α GPIa/IIa

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Platelet Transmission Electron Microscopy

Whole mount Thin section


Mayo Clinic Platelet NGS Panel 69 genes/full exons (ISTH Tier 1)

ABCG5 BLOC1S6 FERMT3 GP9 ITGB3 P2RY12 STIM1

ABCG8 CD36 FLI1 HOXA11 LYST PF4 TBXA2R

ACTN1 CHST14 FLNA HPS1 MASTL PLA2G4A TBXAS1

ADAMTS13 COL3A1 FYB HPS3 MLPH PLAUTHPO

ADRA2A DTNBP1 GATA1 HPS4 MMRN1 PRKACG

ANKRD26 ETV6 GATA2 HPS5 MPL PTGS1 TUBB1

ANO6 F2R GFI1B HPS6 MYH9 RAB27A VIPAS39

ANXA5 F2RL1 GP1BA ITGA2 MYO5A RASGRP2 VPS33B

AP3B1 F2RL2 GP1BB ITGA2B NBEAL2 RGS2 VWF

BLOC1S3 F2RL3 GP6 ITGB1 P2RY1 RUNX1 WAS

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NGS Testing on Both Blood and Skin Fibroblasts

Exon 1 and 2 deletion


Germline RUNX1 mutations: Mayo Clinic Cohort

Modified from DiFilippo, E.C. et al. Leukemia. 2020; 34, 2519–24

1 2 3 4 5 6 7 8

Cavalcante de Andrade Silva M, et al.Cancer Genet. 2018;222-223:32-7.

RUNX1aRUNX1bRUNX1c

Transcripts

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Final Diagnosis

RUNX1 mutation-related inherited platelet disorder

At age of 54, she developed anemia and repeated bone marrow biopsy: hypercellular bone marrow with subtle dysplasia.

Bone marrow HemeOnc NGS testing showed additional mutations:

1. SRSF2: c.284C>A; p.Pro95His (48%)2. TET2: c.3955G>T; p.Glu319* (42%)


2016 WHO

The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia – Arber et al., 2016, 27069254

Myeloid neoplasms with germ line predisposition without a preexisting disorder or organ dysfunction

AML with germ line CEBPA mutationMyeloid neoplasms with germ line DDX41 mutation

Myeloid neoplasms with germ line predisposition and preexisting platelet disordersMyeloid neoplasms with germ line RUNX1 mutationMyeloid neoplasms with germ line ANKRD26 mutationMyeloid neoplasms with germ line ETV6 mutation

Myeloid neoplasms with germ line predisposition and other organ dysfunctionMyeloid neoplasms with germ line GATA2 mutationMyeloid neoplasms associated with BM failure syndromesMyeloid neoplasms associated with telomere biology disordersJMML associated with neurofibromatosis, Noonan syndrome orNoonan syndrome-like disordersMyeloid neoplasms associated with Down syndrome

Classification of myeloid neoplasms with germ line predisposition

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Summary of the Assessment of Case 1










pigmentations




Diagnosis


Clinical AssessmentA 35-year-old female with thrombocytopenia and bleeding diathesis since childhood.

Case 2

2

Age

2230

1516

5

35

Patient History• Ecchymosis and right knee swelling after injury. CBC showed thrombocytopenia • Diagnosed with Evans syndrome (platelets 30 to 40,000/μL), and was treated with prednisone• She was treated with Cytoxan with weekly vincristine for 9 months• Splenectomy due to ongoing low platelets• First pregnancy, delivery was without complication• Underwent second full-term pregnancy with birth. During delivery she had significant vaginal tearing,

and severe bleeding and receive massive transfusion• Since her second pregnancy: she has had many episodes of small hematomas on her hands and

large hematomas on her legs

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Family history


Standard Laboratory TestingCBC CoagulationHemoglobin (g/dL) 9.8 ↓ INR 0.9Hematocrit (%) 36.3 APTT (sec) 31MCV (fL) 89.7 Thrombin time (sec) 17Platelet count (10^9/L) 57 ↓ VWF antigen (IU/dL) 75MPV (fL) NA Fibrinogen (mg/dL) 344White blood cell (10^9/L) 7.9 VWF activity (IU/dL) 74

Platelet Laboratory TestingPFA-100 Epi-cartridge(Sec) ADP-cartridge

236 ↑195 ↑

Platelet aggregation

ArachidonateADPCollagenEpinephrineRistocetin

Normal*Normal*Normal*Normal*Normal*

Platelet glycoproteins Normal GPIIb, IIIa, Ia, Ib-alpha, VI and IX

* Normalized to low platelet counts

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Peripheral blood smear


Platelet Transmission Electron MicroscopyWhole mount Thin section Thin section

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Bone Marrow Biopsy

H&E Reticulin

• Normal chromosome or BCR/ABL1 (FISH)• Negative JAK2 V617F, CALR, MPL mutation analyses


Congenital Myelofibrosis• MPL• JAK2• TPO

• G6B-b• GFI1B• NBEACH2• VPS33b/VIPAS39

• VPS45

Thrombocytosis

Thrombocytopenia

Neutropenia

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Genetic analysis by WES NGS G6B-b (aka MPIG6B or C6orf25) Homozygous c.469G>A p.Gly157Arg

Father: Heterozygous Sister: Homozygous

HGMD: Macrothrombocytopaenia with focal myelofibrosis (DM – CM1816000)

1 2 3 4 65Gene

G6B-b

Ig: Immunoglobulin likeTM: Transmembrane domainITIM: Immunoreceptor tyrosine-based inhibition motifITSM: Immunoreceptor tyrosine-based switch motif

Protein


G6B-b Platelet Biology

Modified from Vogtle T, et al. Elife 2019;8; 1-43.

Extracellular matrix proteins

Shp1/2 Src homology 2 domain-containing protein tyrosine phosphatases 1/2

SFK Src family kinase

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ID HGB(g/dL)PLT

x10^9/L PB smear morphologyG6B-b

genotype Bone marrow findings

4-II-2 5.4 96Large hypogranular

plateletsAbnormal red cells

c.469G>Ap.Gly157Arg Grade 2 myelofibrosis

4-II-3 12.5 97Large hypogranular

plateletsAbnormal red cells

c.469G>Ap.Gly157Arg Grade 2 myelofibrosis

Hofmann I, et al. Blood 2018;132:1399-412.

Thrombocytopenia

Myelofibrosis

Previously Reported Cases


Final Diagnosis

G6B-b mutation-related thrombocytopenia

Treatment options:

Observation, supportive care with platelet transfusionUncertain future progression of myelofibrosis or a myeloid neoplasm

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Congenital Myelofibrosis• MPL• JAK2• TPO

• G6B-b• GFI1B• NBEACH2• VPS33b/VIPAS39

• VPS45

Thrombocytosis

Thrombocytopenia

Neutropenia

Platelet alpha granule deficiency











pigmentations




Diagnosis

Summary of the Assessment of Case 2

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Summary1. Discussed the platelet laboratory testing algorithm

2. Reviewed the platelet esoteric testing

3. Used 2 cases to illustrate our experiences


[email protected]

platelet esoteric testing: when and why...11 ©2020 mfmer | slide-21 ngs testing on both blood and...

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