Super-resolution microscopy as a potential approach to diagnosis of platelet granule disorders

J Thromb Haemost. 2016 Apr;14(4):839-49. doi: 10.1111/jth.13269. Epub 2016 Mar 17.

Abstract

Background: Many platelet functions are dependent on bioactive molecules released from their granules. Deficiencies of these granules in number, shape or content are associated with bleeding. The small size of these granules is such that imaging them for diagnosis has traditionally required electron microscopy. However, recently developed super-resolution microscopes provide sufficient spatial resolution to effectively image platelet granules. When combined with automated image analysis, these methods provide a quantitative, unbiased, rapidly acquired dataset that can readily and reliably reveal differences in platelet granules between individuals.

Objective: To demonstrate the ability of structured illumination microscopy (SIM) to efficiently differentiate between healthy volunteers and three patients with Hermansky-Pudlak syndrome.

Methods: Blood samples were taken from three patients with Hermansky-Pudlak syndrome and seven controls. Patients 1-3 have gene defects in HPS1, HPS6 and HPS5, respectively; all controls were healthy volunteers. Platelet-rich plasma was isolated from blood and the platelets fixed, stained for CD63 and processed for analysis by immunofluorescence microscopy, using a custom-built SIM microscope.

Results: SIM can successfully resolve CD63-positive structures in fixed platelets. A determination of the number of CD63-positive structures per platelet allowed us to conclude that each patient was significantly different from all of the controls with 99% confidence.

Conclusions: A super-resolution imaging approach is effective and rapid in objectively differentiating between patients with a platelet bleeding disorder and healthy volunteers. CD63 is a useful marker for predicting Hermansky-Pudlak syndrome and could be used in the diagnosis of patients suspected of other platelet granule disorders.

Keywords: Hermansky-Pudlak syndrome; diagnostic imaging; platelet storage pool disorder; platelets; structured illumination microscopy; super-resolution.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Albinism, Oculocutaneous / blood*
  • Albinism, Oculocutaneous / diagnosis*
  • Antibodies / chemistry
  • Blood Platelet Disorders / blood
  • Blood Platelet Disorders / diagnosis*
  • Blood Platelet Disorders / immunology*
  • Blood Platelets / cytology
  • Blood Platelets / immunology
  • Codon, Terminator
  • Cytoplasmic Granules / immunology*
  • Frameshift Mutation
  • Gene Deletion
  • Genotype
  • Hemorrhage
  • Hermanski-Pudlak Syndrome / blood*
  • Hermanski-Pudlak Syndrome / genetics
  • Heterozygote
  • Humans
  • Microscopy / methods*
  • Microscopy, Electron
  • Nucleotides
  • Phenotype
  • Platelet Function Tests / methods
  • Platelet-Rich Plasma
  • Tetraspanin 30 / immunology

Substances

  • Antibodies
  • CD63 protein, human
  • Codon, Terminator
  • Nucleotides
  • Tetraspanin 30