1. Validation and assessment of mepacrine testing
in delta storage pool disease: a 3-centre study
Results
Poster
02265
Véronique Latger-Cannard 1,2*, François Mullier 3,4*, Marie Toussaint-Hacquard1, Jean Devignes 1 , Nicolas Bailly 3,
, Jean-Michel Dogné 2, Bernard Chatelain 3, Thomas Lecompte1,2
* Contributed equally
1 CHU Nancy, Service d’hématologie biologique, Nancy, France,2 Centre de Compétence des Pathologies Plaquettaires CCPP, France, 3Hematology Laboratory- Namur Thrombosis and Hemostasis Center (NTHC)-NARILIS, UCL Mont-Godinne, Belgium; 4 Department of Pharmacy –NTHC-NARILIS, University of Namur, Belgium;
Background
Objectives
Delta storage pool disease (dSPD) is probably the most frequent platelet disorder but is still underdiagnosed. Traditional tests to confirm dSPD are transmission electron microscopy (TEM) and lumi-aggregometry. However, though rapid and reliable, TEM requires technical expertise and is only available in highly specialized laboratories. Besides, TEM and lumi-aggregometry are unable to differentiate quantitative from qualitative defects. Mepacrine  testing was first described in 1995 as a useful method to detect dSPD (Wall BJH 1995, Gordon BJH 1995). Despite all methods available, there are nor standardization nor international recommendations related to dSPD diagnosis..
To assess preanalytical, analytical and postanalytical variables in mepacrine test (data not shown)
To provide interinstrument and interlaboratory comparison in mepacrine and CD63 tests
To compare the flow cytometric assay (mepacrine and CD63 ) with Light Transmission Aggregometry (LTA), lumi-aggregometry and clinical outcome in suspicions of dSPD
To propose some modifications of the mepacrin protocol based on this comparison
Methods
After approval by the local ethical committee , this retrospective study included 99 patients with personal or familial history of bleeding or thrombocytopenia. Platelet dense granules were screened by LTA, lumi-aggregometry, mepacrine assay (Wall BJH 1995) and a new CD63 flow cytometry (FCM) test. Patient enrollment started in January 2006 and ended on June 2009.
In addition, bleeding score , familial investigation, platelet count, mean platelet volume (MPV) were recorded for all patients.
For bleeding patients (n=12) flow cytometric of α-granules compounds was also investigated .
CD63: Briefly, CD63 expression was measured by FCM on resting and stimulated platelets. Results are expressed by a CD63 secretion ratio.
Results
Figure 1: Exemple of mepacrine testing in
A) one healthy control B) one dSPD
Table 2: Mechanisms
stimulation
mepacrine
Absence of dense granules
Or  
(lysosomes)
Dense granules
ATP
ATP secretion
Normal dense granule
Empty pool with normal membrane
Nal
Lower number of dense granules 
Exocytosis defect
Mepacrine capture ratio
Mepacrine secretion ratio
CD63 secretion ratio
Nal or 
Fusion without any secretion
Or exocytosis
defect
Table 1: Interpretation of lumiaggregometry, mepacrine test and CD63 patterns in 18 patients with abnormal aggregation pattern
CAPTURE 16μM N=
Lumiaggregometry
Mepacrine capture ratio
Mepacrine secretion ratio
CD63 secretion ratio
Pattern 11
Decreased
Normal or decreased
Decreased number of dense granules 6
Normal
Either Decreased number of dense granules or exocytosis defect 1
Exocytosis defect
Ratio: 3.6 A)
RELEASE 16μM
Ratio: 1.8 B)
CAPTURE 16μM
Ratio: 1.9
Figure 3: Comparison of 2 mepacrin protocols
37 patients showed a pattern in LTA, ATP secretion or mepacrine testing, suggestive of dSPD.
Analysis of mepacrine capture and release, TRAP-induced CD63 platelet membrane expression may allow a mechanistic classification of storage pool defects (Table 1 and 2).
Comparison of 2 protocols in one center showed good agreement (Figure 3). Within assay variation, interindividual variation and interlaboratory comparison are shown in Table 3.
The major changes in the consensus revised protocol are: whole blood instead of PRP, optimization of mepacrine concentration and inclusion of red blood cells as an internal control.
RELEASE 16μM
Ratio: 1.2
Table 3: Within assay variation, interindividual variation and interlaboratory comparison
ND: Not determined
Conclusions
We propose a validated rapid protocol for mepacrin and CD63 testing by FCM which could be widely used to evidence dSPD. Morever, this combination will allow to better understand the physiopathology of this entity.
Disclosure
Contact
Dr Véronique Latger-Cannard: ,
Ph. F. Mullier:
The authors have no relevant conflicts of interest to disclose.