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Mepacrine labelling test and uranaffin cytochemical reaction in human megakaryocytes
Abstract:
5-HT storage organelles were observed by electron microscope analysis in human megakaryocytes. They were less numerous per unit of surface than in platelets. Their number depended on the visualization technique employed. Thus after fixation with calcium-enriched glutaraldehyde a higher number of very opaque organelles was observed than of uranaffin-positive organelles after the cytochemical uranaffin reaction. With conventional electron microscopy deep black granules characteristic of dense bodies were not observed. Fluorescent microscopy showed greenish-yellow granules distributed throughout the whole cytoplasm in 96 +/- 1.4% of normal megakaryocytes incubated with mepacrine. In 85.6 +/- 5%, 5.28 +/- 1.28 granules per 10 microns 2 were observed. With the mepacrine labelling test, 74% of the megakaryocytes of a patient with Hermansky-Pudlak syndrome contained no granules. A similar finding was made in the platelets of the same patient. This suggests that mepacrine also stains the dense bodies in the megakaryocytes and that in the Hermansky-Pudlak syndrome the platelet anomaly is secondary to a megakaryocyte anomaly.
Insights
Human megakaryocytes contain 5-HT storage organelles, visualized with mepacrine fluorescent microscopy. Hermansky-Pudlak syndrome megakaryocytes lack these granules, suggesting platelet anomalies stem from megakaryocyte defects.
Area of Science:
- Hematology
- Cell Biology
- Biochemistry
Background:
- Human megakaryocytes are megakaryocytes, the cells responsible for producing platelets.
- 5-Hydroxytryptamine (5-HT), also known as serotonin, is a neurotransmitter stored in platelet dense granules.
- The precise nature and visualization of 5-HT storage organelles in megakaryocytes remain incompletely understood.
Purpose of the Study:
- To investigate the presence and characteristics of 5-HT storage organelles in human megakaryocytes.
- To compare the number and appearance of these organelles with those in platelets.
- To evaluate the utility of mepacrine as a fluorescent marker for these organelles and assess their presence in Hermansky-Pudlak syndrome.
Main Methods:
- Electron microscopy (conventional, calcium-enriched glutaraldehyde fixation, and uranaffin reaction) was used to visualize organelles.
- Fluorescent microscopy with mepacrine incubation was employed to detect and quantify 5-HT storage organelles.
- Megakaryocytes and platelets from normal individuals and a patient with Hermansky-Pudlak syndrome were analyzed.
Main Results:
- 5-HT storage organelles were observed in human megakaryocytes, though less numerous per unit surface area than in platelets.
- The number of visualized organelles varied significantly with fixation and staining techniques.
- Mepacrine fluorescent microscopy revealed greenish-yellow granules in 96% of normal megakaryocytes, with an average density of 5.28 granules per 10 μm².
- 74% of megakaryocytes from a patient with Hermansky-Pudlak syndrome lacked mepacrine-stained granules, a finding mirrored in their platelets.
Conclusions:
- Mepacrine effectively stains dense bodies in megakaryocytes, similar to its action in platelets.
- The absence of these granules in Hermansky-Pudlak syndrome megakaryocytes suggests that the platelet anomaly in this condition is secondary to a primary megakaryocyte defect.