INHERITED PLATELET DISORDERS LABORATORY MANAGEMENT AND CLINICAL CASES WITH SPECIAL INTEREST ON FLOW

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INHERITED PLATELET DISORDERS LABORATORY
MANAGEMENT AND CLINICAL CASES WITH SPECIAL
INTEREST ON FLOW CYTOMETRYNicole SCHLEGEL, MD,
PhDDirectorBiological Hematology
DepartmentRobert Debré HospitalPARIS-FRANCE
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I. INHERITED PLATELET DISORDERS LABORATORY
MANAGEMENT
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INHERITED PLATELET DISORDERSMAIN CHARACTERISTICS
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ASSESSMENT OF PLATELET FUNCTIONTRANSLOCATION OF
PLATELET GLYCOPROTEINS AND P-SELECTIN DURING
PLATELET ACTIVATION
RESTING
ACTIVATED
Fibrinogen
?
gt 500
50,000
?
? granules
P-selectin
?
GPIIb-IIIa
?
GPIIb-IIIa
GPIV
P-selectin
GPIb/IX/V
?
?
GPIb/IX/V
?
GPIV
ACTIVATION
?
25,000
ACTIVATION - GPIb IX V internalized -
GPIIbIIIa 1) membrane expression increased
2) complex occupied by fibrinogen, v.
Willebrand Factor ... - P-selectin
translocated to the membrane
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INHERITED PLATELET DISORDERSDIAGNOSTIC STRATEGY
(1)
HEMORRHAGIC SYNDROME or SYSTEMATIC
INVESTIGATION ? before surgery ? because
personal or familial past history of
hemorrhage
INITIAL CLINICAL SITUATION
HYPOTHESIS INHERITED PLATELET DISORDER ? Specifi
c platelet studies
HYPOTHESIS v. WILLEBRAND DISEASE ? confirmation
tests
Bleeding Time ? Platelet Numeration N or ?
?
?
APTT N
APTT ?
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INHERITED PLATELET DISORDERSDIAGNOSTIC STRATEGY
(2)
SPECIFIC PLATELET STUDIES
? morphology size, volume
ORIENTATION 1
? aggregation tests
ORIENTATION 2
?? biochemistry immunology flow cytometry
molecular genetic electron microscopy
CONFIRMATION IDENTIFICATION CHARACTERIZATION
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CHARACTERIZATION OF THE PLATELET MEMBRANE
STRUCTURES AND OF THE PLATELET FUNCTION BY FLOW
CYTOMETRY
? PRINCIPLE OF THE TESTS ? MONOCLONAL
ANTIBODIES ? GUIDELINES - material and
reagents - methods - data evaluation - quality
control ???ref. Schmitz et al, Thromb Haemost,
1998, 79, 885
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II. INHERITED PLATELET DISORDERSCLINICAL CASES
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PATIENTS AND FAMILIES - CONTROLS
Patients and families 17 TOTAL
27 Controls (adults) 10
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PATIENTS AND FAMILIES
? Glanzmann Thrombasthenia 7 Type I -
patient 1 - heterozygotes 3 5 -
family member (not mutated) 1 Type II -
patient 1 - daughter ? 1 ? Gray
platelet syndrome-like 3 -
patient 1 - parents 2 ? Fechtner
syndrome 1 ? X-linked familial
thrombocytopenia 5 - patients 3 -
female carriers 2 ? Increased bleeding time of
unknown origin 1
2
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METHODS
QUANTITATION OF - platelets glycoproteins
GPIIbIIIa, GPIIIa, GPIb, GPIb?, GPIX, GPV -
P-selectin (GMP140) in RESTING and ACTIVATED
PLATELETS using Kits Platelet GP
(Biocytex) and Cytoquant (Diagnostica Stago)
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NORMAL VALUES M ab BOUND/PLATELET
n 10
Resting TRAP
Platelets GPIIbIIIa 38,975-58,483 57,580-111,5
55 GPIIIa 39,703-59,313 58,801-126,007 GPIb
26,366-42,113 15,472-33,310 GMP140 lt
523 6,821-15,879
Kits Platelet GP, Cytoquant
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GLANZMANN THROMBASTHENIA
? Quantitative and/or qualitative inherited
deficiency in GPIIbIIIa ? 3 Types I, II and
variants ? More than 50 mutations
3 FAMILIES STUDIED
??TYPE I
? TYPE II
??? TYPE I ? 1 male adult ? heterozygote ?
gipsy mutation G9263?A splicing site intron 15
GPIIb gene ?STOP CODON
normal ? heterozygote ?
? 1 STOP CODON EXON 4 GPIIIa ? other ?
mutation under study
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GRAY PLATELET SYNDROME LIKE
Platelet abnormalities as a manifestation of the
ARC or ARCC-NDI Syndrome (Arthrogryposis, Renal
Fanconi, Cholestasis, or ARC Cerebral
Nephrogenic Diabetes Insipidus Coleman et al,
Am J Med Genet, 1997, 72, 335)
In some cases, large platelets, lacking a
granules and with low bTG, PF4 and TSP Autosomal
recessive inheritance
The family studied
Ivory Coast Parents consanguineous
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FECHTNER SYNDROME
Hereditary thrombocytopenia with giant platelets
and inclusion bodies in the leucocytes
(Different from May-Hegglin and Sebastian
platelet Syndrome)
? MODE OF INHERITANCE ? AUTOSOMAL DOMINANT ?
PLATELETS - ? NUMBER, ? SIZE, ? VOLUME,
????function (collagen) - moderate
bleeding tendency, variable bleeding time - ?
a granules, defective microtubule system -
MEMBRANE ? ? GRANULOCYTES - blue-staining
inclusion bodies - function generally not
impaired - ? myeloperoxidase ? PATHOLOGICAL
ASSOCIATIONS - Alport variant - congenital
cataract - neurosensorial deafness
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X-LINKED THROMBOCYTOPENIA
??Inherited platelet disorder defined by varying
degrees of thrombocytopenia and low platelet
volume ? Allelic variant of Wiskott-Aldrich
Syndrome (WAS)
Study of one family, with Thr45 ? Met mutation
within the exon 2 of the WAS Protein (WASP)
encoding gene de Saint Basile G, Schlegel
N, Caniglia M, Le Deist F, Kaplan C,
Lecompte T, Piller F, Fischer A, Griscelli C. Ann
Hematol, 1991, 63, 107 de Saint Basile
G, Dufourcq Lagehouse R, Lambert N, Schwartz K,
Le Mareck B, Odent S, Schlegel N and Fischer
A. J Pediatr, 1996, 129, 56
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X-LINKED THROMBOCYTOPENIA
Anti Platelet GPIIbIIIa
GPIb GMP140 antibodies R
A R A R A IgG/plat
Coombs specificity (MAIPA) III 1 ? 1760 ?
Auto 27,044 41,807 15,300 15,463
lt500 1,433 anti-IIbIIIa III 2 ?
10795 ? Auto 12,148 15,044 8,578
7,694 lt500 lt500 anti-IIbIIIa III
3 ? 4150 ? No Auto 15,012 35,765
14,338 18,433 lt500 713
anti-IIbIIIa CONTROL 60,757 63,034 38,261
15,577 lt500 2,970
R Resting platelets INTS (C. Kaplan) A
Activated platelets by TRAP Number of M
ab bound/plat
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X-LINKED THROMBOCYTOPENIA
(A)
(A)
(B)
(B)
III.1
III.2
10
10
5
5
(B)
(A)
III.3
(A) M ab bound/platelet (x103)
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resting state
TRAP activation
GPIIbIIIa
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GPIb
GMP140
(B) IgG/platelet (x103)
Auto anti IIbIIIa YES
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DISCUSSION
??Preanalytical recommendations ? Different
platelet activators ? different effects ?
Antibodies panel ? specific diagnosis ? Platelet
bound antibodies ? Normal values/age ? Position
of flow cytometry in the diagnosis of
inherited platelet disorders
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CONCLUSION (1)
? FLow cytometry is an interesting tool as an
initial step for the diagnosis of inherited
platelet disorders ? Results obtained with
Platelet GP or Cytoquant were well correlated
with the clinical cases and with the results
obtained before using other methods
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CONCLUSION (2)
? These methodologies can be applied to other
platelet studies as acquired dysfunction of
platelets or monitoring of therapeutical effects
on platelets ? Multicentric studies will be
helpful to standardize operation procedures and
to better characterize the different types of
platelet disorders
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Marie-Françoise HURTAUD-ROUX Lydia
MAISONNEUVE Biological Hematology Claire
GOULVESTRE Department Annick
RAMBALDINI Sylvie LE GAC Biological
Immunology Catherine PIGNAL Department
Robert Debré Hospital - PARIS