(P-HC-13) Validation of Platelet Granule Storage and Release Testing by Flow Cytometry: A New Assay Offered by the Vitalant Coagulation Laboratory

Various methodologies have been utilized in the clinical laboratory for detection of platelet granule storage and release issues. These include light transmission aggregometry (LTA), lumiaggregometry, electron microscopy (EM), and adenine nucleotide detection. Each methodology has advantages and disadvantages when it comes to analytical and clinical sensitivity/specificity, as well as turn-around time. The Vitalant Coagulation Laboratory sought to develop a test that can detect platelet granule storage and release issues, assess platelet granule issues in thrombocytopenic patients, and offer a 24-hour turn-around time.

Study

Design/Methods:

The following antibodies were chosen for test development: CD61 (platelet surface marker), CD63 (dense granule marker), and CD62P (alpha granule marker).  The compound quinacrine dihydrochloride (mepacrine), which binds adenine nucleotides and fluoresces in the FITC channel, was chosen to quantify adenine nucleotide storage. Platelets were stained with these markers and stimulated with TRAP-6-amide and adenosine disphosphate (ADP) for 30 minutes. Dense granule adenine nucleotide storage (median fluorescence intensity, MFI) was quantified in resting and stimulated platelets. Dense granule release was calculated by dividing the MFI of resting platelets by MFI of stimulated platelets. Alpha granule release was quantified by the MFI of CD62P in stimulated platelets.

Results/Findings:

Reference ranges were set for each parameter to encompass >97% of normal donors. Precision was calculated with three separate donors across three different days. Total precision for adenine nucleotide storage in resting platelets was excellent, with a CV of 6.9%. Release parameters were more variable. Accuracy was performed with at least 20 samples, which were a combination of donors with genetic disorders well described to have delta storage pool issues, as well as samples with abnormal platelet aggregations and available adenine nucleotide results. A patient with Hermansky Pudlak syndrome and no delta granules on EM had a severe adenine nucleotide deficiency detected on flow cytometry (18.8% normalized MFI, % Norm MFI). A patient with Jacobsen syndrome also displayed a moderate adenine nucleotide storage deficiency of 43.5 % Norm MFI. Stability studies demonstrated that storage and release parameters can be reliably assessed only on the day of blood draw and that adenine nucleotide storage is the only parameter that can be assessed reliably in thrombocytopenic patients.

Conclusions:

The Vitalant Coagulation Laboratory has developed a highly sensitive clinical test that can reliably assess both platelet granule storage and release parameters and offers a less than 24-hour turn-around time (TAT) as compared to TAT of several days or weeks for adenine nucleotide and electron microscopy testing.