Abstract Number: PB1673
Meeting: ISTH 2020 Congress
Background: Thrombocytopenia is one of the most challenging limitations for lumi-aggregometry assays and even more if platelet size is increase. Interpretation in such situations requires deep knowledge and some cases need complementary assays to get a conclusion. Flow cytometry (FC) assays assess platelet behavior individually and in combination with lumi-aggregometry, provides a better approach about platelet function. Mepacrine uptake/release assay by FC (Mep u/r) is used to characterized storage pool disease (SPD) and evaluates granule content release.
Aims: To evaluate some technical aspects of Mep u/r and the contribution along with lumi-aggregometry in functional characterization of patients with thrombocytopenia.
Methods: We evaluate a normal group (NG, n: 37), a SPD group (SPD, n: 4) as pathologic control, and 14 patients with thrombocytopenia (TP1-14; platelet count < 150.109/L). Mep u/r was carry out as Cai et al. 2016, using platelet rich plasma, Trap-6 60uM as agonist inducing maximum degranulation. Platelet count dependency was tested in 5 individuals from NG at 50-100-300.109platelets/L. ATP release by lumi-aggregometry (ATPr) were performed using ADP, ADR, arachidonic acid and collagen. TP group results was interpreted according to platelet count.
Results: Mep u/r was independent of the platelet count in the evaluated range (table 1). SPD group had normal platelet count, absent ATPr and low Mep u/r. ATPr in PT group was normal in PT1-11 and correlates well with Mep u/r except in 2 cases (PT10-11). Degranulation of PT10-11 was reevaluated with PMA on FC showing normal results. PT12-14 had decrease ATPr or its inconclusive due to big platelet size and were diagnosed as SPD through abnormal Mep u/r (table 2).
Conclusions: Mep u/r can be used on thrombocytopenia and correlates well with ATPr. PT10-11 showed abnormal Mep u/r with TRAP-6 exclusively, suggesting some alteration in its signaling pathway. In such situations it may be necessary use more than one agonist.
|Uptake||Release ( TRAP-6 60μM)|
|( .109/L)||log (mep/aut)/log FSCH||log (mep/mep-r)|
|0.55±0.09 (mean ±2SD)||0.55±0.17 (mean± 2SD)|
|ANOVA||p< 0.05 (ns)||p< 0.05 (ns)|
|aut= platelets MFI without mepacrine ; mep= resting platelets MFI with mepacrine;FSCH= mean of frontal scattering histogram; mep-r= platelet MFI after TRAP-6 stimulation|
[Table 1_ Comparation between means]
|Platelet count (range= 150-450) (.109/L)||ATPr* (according to platelet count)||Mepacrine Uptake (range= 0.46-0.64)||Mepacrine Release (range= 0.38-0.72)|
|NG (n= 37)||150-490**||Normal||0.46-0.53**||0.46-0.70**|
|SPD (n=4)||150-338**||Absent with all agonist||0.19-0.33**||0.04-0.24**|
|PT12||108||Absent with all agonits||0.28||0.12|
|PT13||82||Absent with all agonist||0.30||0.18|
|*AA: 0,5-1 mM; ADP: 2,5-5 uM; ADR: 10-50 uM: COL1 : 1- 8µg/ml; **:min-max range|
[Table 2_ Lumy-Aggregometry and FC results]
To cite this abstract in AMA style:Asensio M, Alberto MF, Bermejo E, Kinen A, Agazzoni M, Romero ML, Sanchez-Luceros A. Mepacrine Uptake & Release Assay in Thrombocytopenic Patients [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/mepacrine-uptake-release-assay-in-thrombocytopenic-patients/. Accessed November 28, 2023.
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