Phenotyping platelets from patients with inherited platelet disorders by immunofluorescence microscopy

K. Althaus1, C. Blumentritt1, U. Strobel1, T. Bakchoul2, A. Greinacher1 (1Greifswald, Germany, 2Tübingen, Germany)

Platelets - Physiology and Disorders of platelet number and function
Date: 17.02.2017,
Time: 17:15 - 18:15

Objective: Hereditary platelet disorders are more frequent than previously anticipated. Progress in hematology has identified many hereditary causes for thrombocytopenia and/or platelet function defects, but diagnosis is still challenging. It requires fresh blood and laboratory techniques available only in few specialized centers. Further, required blood volumes are usually prohibitive for investigating young children. Next generation sequencing techniques might allow characterization of the underlying genetic defect. However, it remains challenging to correlate the numerous variants within the genome to the phenotype, especially if the underlying platelet defects are poorly characterized. On the other hand, many hereditary platelet defects are associated with characteristic changes in the distribution of specific protein.

Methods: We developed a method to narrow down or confirm the diagnosis of many hereditary platelet disorders. Standard blood smears are prepared by the treating physician and are then shipped by regular mail. The use of specific fixation and permeabilization methods, followed by staining with specific antibodies, allows us to “phenotype” platelets by immunofluorescence microscopy.

Results: Assessing blood smears of 1150 patients referred to our laboratory with unclear thrombocytopenia or platelet function disorders, we achieved the diagnosis in 260 (23%) patients: MYH9-disorders, 145 patients; Bernard Soulier syndrome (BSS), 25 patients; gray platelet syndrome, 2 patients; GFI-1b mutation, 3 patients; ß1-tubulin defects, 10 patients; Wiscott-Aldrich syndrome (WAS), 1 patient; Glanzmann thrombasthenia, 16 patients; alpha storage pool defects, 25 patients; delta storage pool defects, 33 patients. Diagnostic sensitivity and specificity of the method was high for MYH9-disorders/related disease, biallelic Bernard-Soulier syndrome, Glanzmann thrombasthenia, and gray platelet syndrome.

Conclusion: Immunofluorescence microscopy requires minimal blood volumes and allows broad access to specialized laboratories. This morphological technique supports the essential phenotyping of platelets to interpret results of NGS panels for platelet disorders.