Flow Cytometric Evaluation of Platelet Disorders

Dong Chen MD PhDSpecial Coagulation Laboratory

Division of HematopathologyMayo Clinic

DISCLOSURE

•

Relevant Financial Relationship(s)None

•

Off Label UsageNone

Objectives

•

The Concept of Flow Cytometry

•

Applications of Flow Cytometry in Platelet Function Testing

•

Applications of Flow Cytometry in Other Coagulation Testing

The Concept of Flow Cytometry

Platelet Biology-

Focus on the Surface

Platelet “Surface”

Deficiencies

Nurden

2008 Thromb

Haemost

2008, 99: 253

Lou Herzenberg -

1969

-

sorter based on fluorescence (arc lamp) built after working with one of Kamentsky’s

RCS systems where they built an instrument they called the Fluorescence Activated Cell Sorter (FACS)

Kamentsky

-

Bio/Physics Systems -

1970

commercial cytometer

1974

–

present commercialization and further development

BD FACSCalibur

System BD FACS Canto

History of Flow Cytometry

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Flow Cytometry in General

Platelet Flow Cytometry

Diagnosing Platelet Abnormalities by Flow Cytometry

Disease Deficiency Testing by Flow CytometryGlycoprotein (GP) Abnormality:Glanzmann thrombastheniaBernard-Soulier syndromePlatelet-type VWDCollagen receptor deficiencyWiskott-Aldrich syndrome

GP IIb-IIIaGP Ib-V-IXGP IbGP Ia-IIa

& GP IVCD43

Thromb

Res.

1992;67(4):457-66.Haematologica.

1989;74:39-44.

Thromb

Res.

1992 Jan 15;65(2):165-76.Flow on lymphocytes

Abnormal Granules:Storage pool deficiencyGray platelet syndrome

Delta-granuleAlpha-granule

Br J Haematol. 1995 Jan;89(1):117-23. J Clin

Invest. 1991 Mar;87(3):919-29.

Abnormal Signal Transduction:Platelet receptor defects (congenital or acquired)Defects in arachidonic acid metabolismDefects in PLC and Calcium responsiveness

P2Y12 ADP receptor (VASP)COXPLC and Ca2+

Thromb

Haemost. 1999 Sep;82(3):1145-52.

Cytometry B Clin

Cytom. 2008 Mar;74(2):110-7Cytometry. 1988 Mar;9(2):138-42.

Abnormal Procoagulant ActivityScott syndromeMicroparticle

ProcoagulantProcoagulant

Blood. 1993 May 15;81(10):2554-65. Thromb

Res. 1996 Jun 15;82(6):509-22.

Other Acquired ConditionsHITITP

Anti-Heparin/PF4Platelet Binding Ig

Cytometry B Clin

Cytom. 2004 Mar;58(1):32-8. Am J Clin

Pathol. 1987 Apr;87(4):518-22.

Principles of Flow Cytometry

YYYY

YY

Platelet Flow Cytometry Tests

•

Glycoprotein Profile

•

Platelet Function Analysis

YY YY

YYYY

YY

YY

YYYY

YYYY

YY

YY

YY

YY

YY

YYYY

YY

Platelet Glycoprotein Profiles

Early Examples in Literature

Unaffected Carrier Patient

Platelet Function Analysis by Flow Cytometry

YY

YY YY YY YY YYYY

YYYYYYYY

P-Selectin

Activated GP IIb-IIaYYYY

YYYY

Anti-P-Selectin

PAC-1 Antibody

Activation

GP IIb-IIa

P-SelectinPS PS

Annexin V

YYYYPAC-1 Antibody Anti-P-Selectin Annexin

V

General ProcedureWhole Blood Collection

Dilute (and Treat with Stimulus)

Stain with Fluorescent Conjugated Ab

Dilute and Fix

Applications of Flow Cytometry in Platelet Testing

Applications of Flow Cytometry to the Study of Platelets

•

Specific Glycoprotein Deficiency–

Bernard-Soulier Syndrome–

Glanzmann Thrombasthenia–

Collagen Receptor Deficiency

•

Platelet Other Deficiency–

Activation deficiencies to –

Procoagulant Activity

•

Monitoring of Antiplatelet Agents–

Thienopyridine (clopidogrel)–

GPIIb-IIIa Antagonists (Abciximab)

Case 1•

36-year-old man for evaluation of a bleeding disorder. •

He bled for an entire month after his wisdom teeth extraction. •

At that time, his platelet count was 80,000/ul. •

Bleeding history:–

No bleeding after tonsillectomy at age of 3–

No bleeding after scalp laceration at age of 10•

Family History:–

Paternal grandfather had bleeding problems following removal of skin lesions

–

Father had no significant bleeding history–

Mother had a history of abdominal bleeding during pregnancy –

One brother died at the age of 10 from intracerebral bleeding following a playground accident.

Coagulation Testing•

Normal CBC except for thrombocytopenia (80,000 X109/L)•

Giant Platelets on peripheral blood smear•

Normal PT, APTT, TT, VWF, FVIII, FIX, and FXIII•

Bleeding Time: 9

min (1.5-8 min)•

PFA-Epi: 194s (70-165); -ADP: >300 (50-115)•

Platelet Aggregation:–

Arachidonate N–

ADP N–

Epi N–

Collagen N

–

Ristocetin (1.0 and1.5 mg/mL) ↓

Flow Cytometry Studies

100 101 102 103 104Forward Scatter

100 101 102 103 104CD61 FITC

100 101 102 103 104CD42b PE

100 101 102 103 104CD42a FITC

Size

GPIIIa GPIX

GPIbα

Control: Green Patient: Red

Even

tsEv

ents

Even

tsEv

ents

Diagnosis: Bernard-Soulier Syndrome

•

Autosomal Recessive •

First described in 1948

–

Bernard J, Soulier JP. Sur un nouvelle variete' de dystrophie

thrombocytaire

hemorragipare

congenitale. Sem

Hosp Paris 1948;24:3217.

•

Prevalence = 1/million

Common Clinical Features

•

Epistaxis

70%•

Ecchymoses

58%•

Menometrorrhagia

44%•

Gingival Hemorrhage 42%•

GI bleeding 22%•

Posttraumatic Bleeding 13%•

Hematuria

7%•

Cerebral Hemorrhage 4%•

Retinal Hemorrhage 2%

Lopez JA et al. Bernard-Soulier syndrome, Blood 91:4397

Bleeding symptoms: usually evident shortly after birth or in early childhood.

13

Nurden

AT, Nature 255, 720-722

Drouin

J, Blood 1988, 72:1086

Bernard-Soulier Syndrome- GPIb-V-IX Deficiency

NL BSS

Patholophysiology

of BSS

Wintrobe’s Clinical Hematology 2009 Kunicki TJ Page: 1361.

Simon D. et al Haemophilia Vol. 14, 6 Pages: 1240-1249

Mutations of GPIb-V-IX Complex

Platelet 2007 2nd

Edition-page 1034Inherited Disorders of Platelet FunctionAlan T. Nurden

and Paquita

Nurden

Features of BSS Hemorrhagic Diathesis

•

Thrombocytopenia•

Giant Platelets (>10 µm George JN Br J Haematol

1981;48:459–467.

)•

↓

Ristocetin induced PLT Aggregation•

↓

or absent GPIb-V-IX complex•

Classification:–

Type 1: Quantitative/Qualitative–

Type 2: Normal platelet count and size (Kenny D. et al. 1998 Blood 92: 175)

Treatment

•

Platelet transfusion for severe bleeding•

Desmopressin•

Recombinant factor VII•

Antifibrinolytic agents

Trivia for BSS

•

Rare BSS variant my have normal platelet count and size.

•

Patients with DiGeorge syndrome may have missing GPIb

beta.

•

Degrees of GPIb

and IX deficiency do not appear to correlate with bleeding severity.

•

GPV is not a essential component and its abnormalities do not cause BSS.

Flow Cytometry Studies for Mailed-in Samples

Patient: 34y/o female with significant bleeding history

Mild thrombocytopenia

Normal Plasmatic Coagulation Studies

PFA 100: abnormalPlatelet Aggregation Test:

Arachidonic Acid: AbnormalADP: AbnormalEpi: AbnormalCollagen: AbnormalRistocetin: Decreased

Case 2

Flow Cytometry-Glycoprotein Profile

GPIIb

GPIbα GPIX

GPIIIa

Resting Platelets: BlackADP (0.1mM): Green

Normal Control Patient

Flow Cytometry-Activity Panel

P-selectin

Pac-1

Diagnosis: Glanzmann Thrombasthenia

•

First described in 1918 by Glanzmann E, (J kinderkranken

1918,88:113)

•

Autosomal recessive•

Clinical Description–

Homozygous: •

Minimal bruising to severe hemorrhages•

Bleeding symptoms manifest rapidly after birth•

Bleeding tendency improves with age–

Heterozygous: •

No bleeding

•

GPIIb/IIIa deficiency:

Diagnosis: Glanzmann Thrombasthenia

16

24

Nurden

AT, Nature 255, 720-722

Normal Donor GT Patient

Diagnostic Methods for GT•

Clinical/family history •

Peripheral blood•

Routine Coagulation studies•

Platelet Function studies–

PFA-100–

Platelet Aggregation:•

Arachidonic acid: ↓•

ADP ↓•

Epi ↓•

Collagen ↓•

Ristocetin NL/ ↓•

Flow cytometry: GPIIb/IIIa ↓ ↓ ↓•

Mutation analysis: best for prenatal and carrier status analyses.

Pathophysiology of GT

Mutations of GPIIb and GPIIIa

Orphanet Journal of Rare Diseases 2006, 1:10

http://sinaicentral.mssm.edu/intranet/rese

arch/glanzmann/menu

Trivia about GT

•

Treatment:–

Platelet transfusion for severe bleeding –

Recombinant FVII (Poon

MC, JTH 2004, 2: 1096) and antifibrinolytic agents (Lisman

T, Blood, 2003, 101:1864)

•

Acquired Conditions:–

Acute promyelocytic leukemia (Chen Y. etal

Thromb

Haemost

1989, 62:176)

–

ITP or other autoimmune conditions (Tholouli

E, Br J Haematol

2004, 127:209)

•

Disease Severity: The Unresolved issue.–

Broad spectrum–

Bleeding Severity does not correlate with degrees of GPIIb/IIIa deficiency

Our Experience of Platelet Function Analysis by Flow Cytometry

2006-presentDisorders Cases Number

•

BSS: 4•

GT:

5•

GP Ia-IIa:

2•

Gray Platelet Syndrome 5•

Macrothrombocytopenia NOS

7•

Signal pathway deficiency

3•

Other Disorders

8•

Normal 55

•

Total 89

Problems of Current Platelet Flow Cytometry Practice

•

Lack of Standard Operation–

Pre-analytical: Sample tubes (Citrate and ACD)–

Reagents: antibodies and fluorochromes–

Lack of standarized fluorescent quantification–

SOP

•

Lack of Normal Range–

Due to lack of standarized quantification guidelines–

Age, gender, concomitant conditions

•

Esoteric Practice–

High Complexity: Mailed in specimen

Standardization of Platelet Flow Cytometry Studies

•

Sample collection: Citrate or ACD•

Panels:–

GP screen

–

GP quantification

System Description•

Glycoprotein screening assays:•

-

by mean fluorescent intensity •

-

Whole blood sample in citrate (3.2%) or ACD tubes (A or B)•

-

Conjugated antibodies to •

CD41 (GPIIb) •

CD61(GPIIIa) •

CD42a (GPIX)•

CD 42b (GPIba)•

CD49a (IaIIa)•

-Gated with Forward and Side light scatter histogram.•

-The mean fluorescent equivalences (MFE) are calculated from a standard curve of calibrator beads.

100 101 102 103 104FL1-H

80827.001

M1

M2

M3

M4

M5

M6

Histogram Statistics

Marker Left, Right Events % Gated % Total Mean Geo Mean CV Median Peak ChAll 1, 9910 29984 100.00 70.65 1202.12 254.14 129.20 261.80 4410M1 1, 14 4985 16.63 11.75 3.84 3.46 45.38 3.55 4M2 52, 136 4846 16.16 11.42 81.07 80.59 10.92 80.58 82M3 136, 400 5104 17.02 12.03 231.17 230.55 7.27 230.82 237M4 400, 1165 4979 16.61 11.73 606.19 604.88 6.67 609.76 615M5 1186, 3051 4828 16.10 11.38 2010.99 2008.62 4.73 2035.14 2072M6 3051, 6264 5029 16.77 11.85 4294.34 4290.19 4.34 4332.30 4410

File: 80827.001 Log Data Units: Linear ValuesSample ID: RCP calib Patient ID: Tube: Untitled Panel: Untitled Acquisition Tube ListAcquisition Date: 27-Aug-08 Gate: G1Gated Events: 29984 Total Events: 42438X Parameter: FL1-H (Log)

Example

Example of MFE calculation

Quantitative glycoprotein screen test by Biocytex: -

Whole blood in citrate or ACD tube. -

Anti-CD61 (IIIa), -CD42a (GPIX) and -CD49a (GPIa/IIa) antibodies, and then stained with conjugated secondary antibody.

*The MFI will be compared with a standard curve derived from a standard primary antibody coated beads.

*Copy numbers of glycoproteins will extrapolated from the standard curve.

Platelet

= GP=Mouse Anti-GP ab= FITC-anti-mouse ab

= mouse IgGof known molecules/bead

bead bead bead bead

Add anti-mouse IgG-Fluorescein conjugate

in one tube

in another tube

The beads give 4 peaks of fluorescence.One creates a standard curve of MFI vs.molecules/platelet(bead). GP/platelet isread off of the curve

100 101 102 103 104anti-Mouse IgG FITC

80502.005

M1

M2

M3

M4

Biocytex

GP Screen

100 101 102 103 104anti-Mouse IgG FITC

80502.005

M1

M2

M3

M4

Histogram Statistics

Marker Left, Right Events % Gated % Tota Mean Geo Mean CV Median Peak ChAll 1, 9910 20000 100.00 100.00 1147.32 377.02 98.77 873.79 1M1 1, 155 4813 24.07 24.07 30.75 18.41 87.14 23.71 45M2 155, 723 4757 23.79 23.79 380.52 370.27 23.19 375.16 352M3 723, 1928 4948 24.74 24.74 1209.69 1195.38 15.36 1207.90 1197M4 1928, 4532 5207 26.04 26.04 2878.95 2859.57 11.72 2864.38 2838

File: 80502.005 Log Data Units: Linear ValuesSample ID: GP Screen Patient ID: bead mix + anti-MIgG FITCTube: Untitled Panel: Untitled Acquisition Tube ListAcquisition Date: 02-May-08 Gate: No GateGated Events: 20000 Total Events: 20000X Parameter: anti-Mouse IgG FITC (Log)

Standard curve

bead peak MCF Mur IgG/bead

A 18.7 490

B 380.71 13000

C 1243.53 38000

D 2907.62 96000

GP screen standard curve

y = 32.915x - 572.58R2 = 0.9986

0100002000030000400005000060000700008000090000

100000

0 1000 2000 3000

MCFM

ur Ig

G/b

ead

5/3/2008

6-010-15, post-surgery GP screen

sample marker MCF GP sites/platelet normal range (X103)

T2 GPIIIa 1413.25 45,944.1 53±12

T3 GPIbα 972.46 31,435.6 38±11

T4 GPIa 126.9 3,604.3 5±2.8

A B C Dco

unts

200

100

50

Example

Applications of Flow Cytometry in Other Coagulation Testing

VWF Ristocetin (VWF:RCo) Activity by Flow Cytometry

•

-Insensitive to Type 2 VWD–

VWF:RCo/Ag

ratio <0.5-0.7* (NL= 1.0±0.26)

•

-High Coefficient of Variation (15-30%)**–

No calibrated reagent platelets–

Complicated procedure–

Large reaction volume (1mL)–

Unreliable when VWF:Ag <15-20 IU/dL–

Labor intensive

•

* Hillery

CA

et al. Blood 1998; James PD

et al. JTH 2006; Eikenboom

J

et al. JTH 2006; Favaloro EJ

et al. Haemophilia

2004; Federici AB

et al. Thromb

haemost2000; Laffan

M

et al. Haemophilia

2004 •

** Steve Kitchen

et al. Semin

Throm

Hemost

2006

Problems of Current VWF:RCo

Assays by Platelet Aggregation /Aggregometry Method

VWF: RCo

Activity by Flow Cytometry

Chen et al. JTH Volume 6, Number 2, February 2008 , pp. 323-330

VWF:Ristocetin

Testing Procedure

0.5 μl plasma

Validation of VWF:RCo

by Flow Cytometry Method

AggregationCut off

Flow Cut off

Advantages of VWF:RCo

by Flow Cytometry Method

Flow Cytometry Aggregation/Aggregometry

VWF:RCo/Ag ratio for Type 2 VWD

0.5 (more sensitive)More specific

0.5-0.7 (less sensitive)Less specific

Patient Sample Volume

1-2 µL 200 µL

Reagent Platelets1 unit Platelet

Calibrated (fixed)2500 tests (12 months)

Donor variation (fresh)80 tests (Daily)

Linear RangeLowest VWF level

3~150 IU/dL0.1 IU/dL (increase sample volume)

12.5-100 IU/dL12.5 IU/dL

CV% 5-10% 15-30%

Complexity More complexed Complex

Automation Yes (with autoloader) No

Summary

•

The Concept of Flow Cytometry

•

Applications of Flow Cytometry in Platelet Function Testing

•

Applications of Flow Cytometry in Other Coagulation Testing

Acknowledgments

•

Dr. Whyte G. Owen•

Dr. William L. Nichols

•

Dr. Rajiv K. Pruthi•

Dr. John A. Heit

•

Randall S. Miller–

Flow cytometry specialist