Flow Cytometric Evaluation of Platelet Disorders Dong Chen MD PhD Special Coagulation Laboratory Division of Hematopathology Mayo Clinic
DISCLOSURE Relevant Financial Relationship(s) None Off Label Usage None
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
History of Flow Cytometry 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
Publications 0 2000 4000 6000 8000 10000 12000 1975-1976 1976-1977 1977-1978 1978-1979 1979-1980 1980-1981 1981-1982 1982-1983 1983-1984 1984-1985 1985-1986 1986-1987 1987-1988 1988-1989 1989-1990 1990-1991 1991-1992 1992-1993 1993-1994 1994-1995 1995-1996 1996-1997 1997-1998 1998-1999 1999-2000 2000-2001 2001-2002 0 2000 4000 6000 8000 10000 12000 1975-1976 1976-1977 1977-1978 1978-1979 1979-1980 1980-1981 1981-1982 1982-1983 1983-1984 1984-1985 1985-1986 1986-1987 1987-1988 1988-1989 1989-1990 1990-1991 1991-1992 1992-1993 1993-1994 1994-1995 1995-1996 1996-1997 1997-1998 1998-1999 1999-2000 2000-2001 2001-2002 Publications 0 200 400 600 800 1000 1200 1400 1600 1800 2000 Before 1979 1979-1984 1985-1990 1991-1996 1997-2002 2003-2008 ears Publications Publications Publications 0 200 400 600 800 1000 1200 1400 1600 1800 2000 Before 1979 1979-1984 1985-1990 1991-1996 1997-2002 2003-2008 ears Publications Publications Flow Cytometry in General Platelet Flow Cytometry
Diagnosing Platelet Abnormalities by Flow Cytometry Disease Deficiency Testing by Flow Cytometry Glycoprotein (GP) Abnormality: Glanzmann thrombasthenia Bernard-Soulier syndrome Platelet-type VWD Collagen receptor deficiency Wiskott-Aldrich syndrome GP IIb-IIIa GP Ib-V-IX GP Ib GP Ia-IIa & GP IV CD43 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 deficiency Gray platelet syndrome Abnormal Signal Transduction: Platelet receptor defects (congenital or acquired) Defects in arachidonic acid metabolism Defects in PLC and Calcium responsiveness Abnormal Procoagulant Activity Scott syndrome Microparticle Other Acquired Conditions HIT ITP Delta-granule Alpha-granule P212 ADP receptor (VASP) COX PLC and Ca 2+ Procoagulant Procoagulant Anti-Heparin/PF4 Platelet Binding Ig Br J Haematol. 1995 Jan;89(1):117-23. J Clin Invest. 1991 Mar;87(3):919-29. Thromb Haemost. 1999 Sep;82(3):1145-52. Cytometry B Clin Cytom. 2008 Mar;74(2):110-7 Cytometry. 1988 Mar;9(2):138-42. Blood. 1993 May 15;81(10):2554-65. Thromb Res. 1996 Jun 15;82(6):509-22. Cytometry B Clin Cytom. 2004 Mar;58(1):32-8. Am J Clin Pathol. 1987 Apr;87(4):518-22.
Principles of Flow Cytometry
Platelet Flow Cytometry Tests Glycoprotein Profile Platelet Function Analysis
Platelet Glycoprotein Profiles
Early Examples in Literature Unaffected Carrier Patient
Platelet Function Analysis by Flow Cytometry PAC-1 Antibody Anti-P-Selectin Annexin V PAC-1 Antibody GP IIb-IIa Activated GP IIb-IIa PS P-Selectin Activation PS Annexin V P-Selectin Anti-P-Selectin
General Procedure Whole 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 X10 9 /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 Events Size Events GPIbα 10 0 10 1 10 2 10 3 10 4 Forward Scatter 10 0 10 1 10 2 10 3 10 4 CD42b PE Events GPIIIa Events GPIX 10 0 10 1 10 2 10 3 10 4 CD61 FITC 10 0 10 1 10 2 10 3 10 4 CD42a FITC Control: Green Patient: Red
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 Bleeding symptoms: usually evident shortly after birth or in early childhood. 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
Bernard-Soulier Syndrome- GPIb-V-IX Deficiency NL BSS 13 Drouin J, Blood 1988, 72:1086 Nurden AT, Nature 255, 720-722
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 2 nd Edition-page 1034 Inherited Disorders of Platelet Function Alan 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
Case 2 Patient: 34y/o female with significant bleeding history Mild thrombocytopenia Normal Plasmatic Coagulation Studies PFA 100: abnormal Platelet Aggregation Test: Arachidonic Acid: Abnormal ADP: Abnormal Epi: Abnormal Collagen: Abnormal Ristocetin: Decreased
Flow Cytometry-Glycoprotein Profile GPIbα GPIX GPIIb GPIIIa
Flow Cytometry-Activity Panel Resting Platelets: Black ADP (0.1mM): Green Normal Control Patient 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
Diagnosis: Glanzmann Thrombasthenia GPIIb/IIIa deficiency: Normal Donor GT Patient 16 24 Nurden AT, Nature 255, 720-722
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 http://sinaicentral.mssm.edu/intranet/rese arch/glanzmann/menu Orphanet Journal of Rare Diseases 2006, 1:10
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. 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-present Disorders 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
Glycoprotein - - - -Gated -The CD41 CD61(GPIIIa) CD42a CD CD49a System Description screening assays: by mean fluorescent intensity Whole blood sample in citrate (3.2%) or ACD tubes (A or B) Conjugated antibodies to (GPIIb) (GPIX) 42b (GPIba) (IaIIa) with Forward and Side light scatter histogram. mean fluorescent equivalences (MFE) are calculated from a standard curve of calibrator beads.
80827.001 Histogram Statistics M4 M5 M6 File: 80827.001 Log Data Units: Linear Values Sample ID: RCP calib Patient ID: Tube: Untitled Panel: Untitled Acquisition Tube List Acquisition Date: 27-Aug-08 Gate: G1 Gated Events: 29984 Total Events: 42438 X Parameter: FL1-H (Log) M2 M3 Marker Left, Right Events % Gated % Total Mean Geo Mean CV Median Peak Ch All 1, 9910 29984 100.00 70.65 1202.12 254.14 129.20 261.80 4410 M1 1, 14 4985 16.63 11.75 3.84 3.46 45.38 3.55 4 M2 52, 136 4846 16.16 11.42 81.07 80.59 10.92 80.58 82 M3 136, 400 5104 17.02 12.03 231.17 230.55 7.27 230.82 237 M4 400, 1165 4979 16.61 11.73 606.19 604.88 6.67 609.76 615 M5 1186, 3051 4828 16.10 11.38 2010.99 2008.62 4.73 2035.14 2072 M6 3051, 6264 5029 16.77 11.85 4294.34 4290.19 4.34 4332.30 4410 M1 10 0 10 1 10 2 10 3 10 4 FL1-H Example of MFE calculation Example
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.
Biocytex GP Screen = GP =Mouse Anti-GP ab = FITC-anti-mouse ab in one tube Platelet Add anti-mouse IgG- Fluorescein conjugate bead bead bead bead 80502.005 in another tube = mouse IgG of known molecules/bead M3 M4 M2 M1 10 0 10 1 10 2 10 3 10 4 anti-mouse IgG FITC The beads give 4 peaks of fluorescence. One creates a standard curve of MFI vs. molecules/platelet(bead). GP/platelet is read off of the curve
200 80502.005 A B C D Standard curve bead peak MCF Mur IgG/bead A 18.7 490 B 380.71 13000 counts 100 M3 M4 C 1243.53 38000 D 2907.62 96000 GP screen standard curve 50 M1 10 0 10 1 10 2 10 3 10 4 anti-mouse IgG FITC M2 Histogram Statistics File: 80502.005 Log Data Units: Linear Values Sample ID: GP Screen Patient ID: bead mix + anti-migg FITC Tube: Untitled Panel: Untitled Acquisition Tube List Acquisition Date: 02-May-08 Gate: No Gate Gated Events: 20000 Total Events: 20000 X Parameter: anti-mouse IgG FITC (Log) Mur IgG/bead 100000 90000 80000 70000 60000 50000 40000 30000 20000 10000 y = 32.915x - 572.58 R 2 = 0.9986 Marker Left, Right Events % Gated % Tota Mean Geo Mean CV Median Peak Ch All 1, 9910 20000 100.00 100.00 1147.32 377.02 98.77 873.79 1 M1 1, 155 4813 24.07 24.07 30.75 18.41 87.14 23.71 45 M2 155, 723 4757 23.79 23.79 380.52 370.27 23.19 375.16 352 M3 723, 1928 4948 24.74 24.74 1209.69 1195.38 15.36 1207.90 1197 M4 1928, 4532 5207 26.04 26.04 2878.95 2859.57 11.72 2864.38 2838 0 0 1000 2000 3000 MCF 5/3/2008 Example 6-010-15, post-surgery GP screen sample marker MCF GP sites/platelet normal range (X10 3 ) T2 GPIIIa 1413.25 45,944.1 53 12 T3 GPIb 972.46 31,435.6 38 11 T4 GPIa 126.9 3,604.3 5 2.8
Applications of Flow Cytometry in Other Coagulation Testing VWF Ristocetin (VWF:RCo) Activity by Flow Cytometry
Problems of Current VWF:RCo Assays by Platelet Aggregation /Aggregometry Method -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
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 Aggregation Cut off Flow Cut off
Advantages of VWF:RCo by Flow Cytometry Method VWF:RCo/Ag ratio for Type 2 VWD Patient Sample Volume Reagent Platelets 1 unit Platelet Linear Range Lowest VWF level Flow Cytometry 0.5 (more sensitive) More specific 1-2 µl 200 µl Calibrated (fixed) 2500 tests (12 months) 3~150 IU/dL 0.1 IU/dL (increase sample volume) Aggregation/Aggregometry 0.5-0.7 (less sensitive) Less specific Donor variation (fresh) 80 tests (Daily) 12.5-100 IU/dL 12.5 IU/dL CV% 5-10% 15-30% Complexity More complexed Complex Automation es (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