Methodological problems of direct bioluminescent ADP assay in platelets and erythrocytes

Girotti, Stefano; Ferri, Elida Nora; Cascione, Maria Loredana; Orlandini, Alessandro; Farina, Luca; Nucci, S; Graci, F. Di; Budini, Rolando

doi:10.1016/0003-2697(91)90547-7

Cited by 5 publications

(3 citation statements)

References 20 publications

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“…Patients were categorized by the results of EM analysis as δ-SPD or within normal limits. Our laboratory has established normal range dense δ-granule values utilizing well-vetted control subjects who met the following criteria: screened by extensive bleeding questionnaires, normal complete blood cell counts, and normal platelet function analysis including platelet aggregation assays, and assessed for an extracted ATP/ADP ratio (< 2.0) via firefly luciferase assay [ 17 , 18 ]. Our normal range of DG/PL is 4 - 6 (4.60 ± 0.31 DG/PL in our lab), consistent with values established in the literature [ 9 , 11 , 19 ].…”

Section: Methodsmentioning

confidence: 99%

Platelet Aggregation Assays Do Not Reliably Diagnose Platelet Delta Granule Storage Pool Deficiency

2021

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Background Patients with platelet dysfunction disorders present with a variety of mucocutaneous bleeding symptoms including easy bruising, frequent epistaxis, bleeding gums upon tooth brushing and for women, heavy menstrual bleeding. Available laboratory assays to evaluate platelet function include the platelet function analyzer (PFA) and in larger centers with coagulation laboratories, light transmission platelet aggregometry (LTA) analyses. Both assays are known to have a number of limitations, especially in the diagnosis of platelet delta granule storage pool deficiency (δ-SPD). δ-SPD is an underdiagnosed condition caused by decreased numbers of platelet dense granules (DGs) and is best diagnosed by electron microscopy (EM). Patients with platelet δ-SPD have a decreased response to low levels of the agonist adenosine diphosphate (ADP) in the second wave of light transmittance with LTA or decreased ADP secretion by fluorescence lumiaggregometry. There are few reports that have evaluated patients with δ-SPD and their respective LTA results. One report published in 1987 described normal LTA assays in 23% of patients with δ-SPD; a more recent report described LTA as having the sensitivity to detect only about 52% of patients with δ-SPD. The purpose of our study was intended to review the LTA and EM results of patients suspected of having a platelet function disorder at our institution for comparison with previously published studies. Methods Our study included 344 patients who had been evaluated by both LTA and whole mount EM. Aggregometry utilized five agonists: ADP, epinephrine, collagen, arachidonic acid, and ristocetin. DGs were enumerated in 100 whole-mounted platelets to determine a mean number of dense granules per platelet (DGs/PL). Results Seventy-seven percent of our patients were found to have δ-SPD (264/344); 68% (179/264) of these subjects had an abnormal platelet LTA. Thirty-two percent (85/264) of our patients had normal LTA results but were found to have δ-SPD with a mean of 2.54 ± 0.15 DG/PL (normal = 4 - 6 DG/PL). Conclusion These data confirm previous reports suggesting the utilization of LTA alone in patients with histories of unexplained bleeding may miss the diagnosis of platelet δ-SPD. It is, therefore, prudent to assess platelet DG number by EM, especially if platelet LTA assessment is normal.

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Section: Methodsmentioning

confidence: 99%

Platelet Aggregation Assays Do Not Reliably Diagnose Platelet Delta Granule Storage Pool Deficiency

2021

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“…The extraction and determination of platelet adenosine nucleotide content were performed by standard methods [ 3 , 35 , 36 , 37 , 38 ]. A 500 µL sample of PRP was mixed with 450 µL of 96% ethanol (EtOH) and 50 µL of 0.1M EDTA, pH 7.4, for 10 s, and centrifuged for 15 min at 14,000× g .…”

Section: Methodsmentioning

confidence: 99%

A Morphometric Analysis of Platelet Dense Granules of Patients with Unexplained Bleeding: A New Entity of Delta-Microgranular Storage Pool Deficiency

Gunning

Raghavan

Calomeni

et al. 2020

JCM

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One thousand and eighty patients, having prolonged bleeding times, frequent epistaxis, menorrhagia or easy bruising or other bleeding manifestations, and excluding those with von Willebrand’s disease, were evaluated for platelet dense granule deficiency. The mean diameter of platelet dense granules was determined for all patients using image analysis. Four hundred and ninety-nine had “classic” dense (delta) granule storage pool deficiency (δ-SPD). Five hundred and eighty-one individuals (53.8%) were found to have a normal mean number of dense granules, but for some of these patients, the dense granules were smaller than for the controls. Of the patients having a normal number of dense granules, 165 (28.4%) were found to have significantly smaller granules than the platelets obtained from the control subjects. Their average granule diameter was 123.35 ± 0.86 nm, that is more than three standard deviations below the mean of the control data. Total δ-granule storage pool volumes (TDGV)/platelet were calculated using these measurements. Individuals with δ-SPD had half the number of granules (2.25 ± 0.04 DG/PL) and storage pool volume (3.88 ± 1.06 × 106 nm3) when compared to our control data (4.64 ± 0.11 DG/PL; 10.79 × 106 nm3 ± 0.42). Individuals having a bleeding history but a normal average of small dense granules had a calculated storage pool volume statistically different than controls and essentially the same storage pool volume as patients with δ-SPD. We have identified a sub-classification of δ-SPD that we have defined as micro-granular storage pool deficiency (δ-MGSPD).

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“…Isolation of human platelets (HP) plasmatic membranes. Human cells were isolated according to modified methods of David and Herion (1972) and Girotti et al (1991). Platelets were collected from 20 healthy subjects and stored at -70 o C. The platelets were unfrozen, vortexed for 10 s and sonicated as described for RBA.…”

Section: Methodsmentioning

confidence: 99%

Fluorometric assay of oleate-activated phospholipase D isoenzyme in membranes of rat nervous tissue and human platelets.

Krzystanek

Trzeciak²,

Krzystanek³

et al. 2010

Acta Biochim Pol

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Phospholipase D plays a key role in the biosynthesis of phosphatidic acid, a second messenger involved in essential cellular processes. Oleate-activated phospholipase D was the first mammalian phospholipase D isoform to be discovered but is the least known. The study was aimed to test a fluorometric method of assessment of oleate-activated phospholipase D activity in different biological materials. The brain cortex of male Wistar rats, cultured rat brain astrocytes, and human platelets were processed to yield plasmatic membranes for experiments. To assess phospholipase D activity the modified fluorometric method was used. Previously, the method was used only to determine H₂O₂. In this enzyme-coupled assay phospholipase D activity is monitored indirectly using 10-acetyl-3,7-dihydroxyphenoxazine. First, phospholipase D cleaves exogenous phosphatidylcholine to yield choline and phosphatidic acid. Second, choline is oxidized by choline oxidase to betaine and H₂O₂. Finally, in the presence of horseradish peroxidase, H₂O₂ reacts with 10-acetyl-3,7-dihydroxyphenoxazine to generate the highly fluorescent product, resorufin. The concentration of resorufin was measured using excitation and emission at 560 nm and 590 nm, respectively. The proposed optimal parameters of the tested assay are 25 µg of rat brain cortex protein, 50 µg of rat brain astrocyte protein, and 50 µg of human platelet protein in a reaction volume of 200 µL, and 2 min enzymatic reaction at 37°C. The fluorometric method may be applied to assay phospholipase D in different biological materials.

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Methodological problems of direct bioluminescent ADP assay in platelets and erythrocytes

Cited by 5 publications

References 20 publications

Platelet Aggregation Assays Do Not Reliably Diagnose Platelet Delta Granule Storage Pool Deficiency

Platelet Aggregation Assays Do Not Reliably Diagnose Platelet Delta Granule Storage Pool Deficiency

A Morphometric Analysis of Platelet Dense Granules of Patients with Unexplained Bleeding: A New Entity of Delta-Microgranular Storage Pool Deficiency

Fluorometric assay of oleate-activated phospholipase D isoenzyme in membranes of rat nervous tissue and human platelets.

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