White, James G.

Abstract Germline mutations in the X-linked hematopoietic transcription factor, GATA-1, have been associated with dyserythropoietic anemia (DEA), macrothrombocytopenia (MTP), and congenital erythropoietic porphyria. Recently, morphological features suggestive of the Gray Platelet Syndrome (GPS) were described in a pedigree with a germline R216Q GATA-1 mutation (Blood106:6a, 2005). The present study has evaluated platelets (Pl) in the electron microscope from members of a previously described pedigree (Blood98:2681–2688, 2001) with GATA-1 G208S, MTP, and dyserythropoiesis without anemia, for whom detailed platelet morphology studies have not been reported. The presence of a hemizygous GATA-1 mutation was confirmed by conventional fluorescent dye chemistry sequencing of DNA from patient peripheral blood mononuclear cells. Their Pl revealed wide variations in size, shape, internal and external structure. Some normal sized and giant Pl contained usual numbers of alpha granules and dense bodies, while most of the large cells were hypogranular. Some contained no membrane systems or organelles. Others were filled with masses of dense tubular system (DTS) channels, and others contained the tubular inclusions found in the Medich Giant Platelets Disorder (Platelets15:345–353, 2004). Many of the hypogranular cells contained small vacuoles that may have been enclosing membranes of alpha granules. Except for the tubular inclusions, all of these structural variations are observed in GPS Pl (Am. J. Pathol.95:445–462, 1979). However, additional striking abnormalities were seen in GATA-1 Pl that are not characteristic of GPS Pl. Many GATA-1 Pl contained unusual, closely associated dense double membranes differing from normal elements of rough endoplasmic reticulum, smooth endoplasmic reticulum, DTS or surface connected open canalicular system (OCS), but resembling channels of the OCS after exposure to EDTA. They were observed previously only in megakaryocytes (Mk) from one family with GATA-1 DEA-MTP (Nat. Genet, 24:266–270, 2000). The dense double membranes were often found in parallel association and, in some examples, isolating areas of cytoplasm. However, the isolated areas were not undergoing autophagic degradation as such areas enclosed by elements of the DTS do in White Platelet Syndrome Pl (Platelets 15:173,2004). Instead, they proved to be another unique feature of GATA-1 platelets, the presence of Pl within Pl. The sequestered cells were usually mature in appearance and often discoid in form with circumferential coils of microtubules. In some GATA-1 Pl there were two Pl within the same cell, and on rare occasions, a Pl within a Pl within a Pl. Pl within Pl have never been observed previously in any human platelet disorder. Another unique feature, related to Pl within Pl, was the frequent Pl to Pl surface attachment of non-activated cells forming large macrothrombocytes with no similarity to aggregates of stimulated normal platelets. Conclusion: The substructural abnormalities of Pl from patients with germline GATA-1 mutations share some overlap with those seen in GPS Pl, but are distinct and unique. The MTP, Pl within Pl and surface Pl to Pl attachments suggest a major defect in the formation and separation of GATA-1 Pl from pro-Pl of the parent Mk, resulting in the MTP characteristic of the disorder.

Abstract Background Platelet dense granule (DG) deficiencies, including Hermansky-Pudlak syndrome, are relatively common and frequently under-diagnosed hereditary platelet hypofunctional disorders. Among various laboratory tests, platelet whole mount (WM) transmission electron microscopy (TEM) is considered the gold standard method for diagnosing DG deficiencies. Nevertheless, platelet DG TEM testing is still largely a research tool and has not been thoroughly validated and standardized as a clinical test. Furthermore, reference ranges (RR) of DG count/platelet (DGC) have not been adequately established. Aim Our goals were to validate and standardize platelet WM TEM testing and establish reference ranges for platelet DGC. Methods Based on previously established methods (Blood, 33:598-606), we first optimized and standardized various pre-analytical, analytical and post-analytical procedures including sample matrix, sample stability, platelet-rich plasma (PRP) preparation, platelet mounting procedure and DG counting criteria. Then whole blood samples in ACD-B tubes were collected from a total of 128 healthy donors (64 males and 64 females). Platelet DGC, percentage of platelets without any DG (empty platelets), platelet count (PC), mean platelet volume (MPV) and immature platelet fraction (IPF) were measured. In addition, platelet aggregometry analyses were performed on citrated PRP samples from the same donors. Standard statistical analyses were employed in this study. Results DGC and platelet ultra-structure were stable when whole blood samples were stored at room temperature (RT) for up to 4 days. Reproducible DGC and adequate preservation of platelet ultrastructure depended on an optimized mounting method. The DG counting criteria were developed based on previous publications and guidance from Dr. James G. White. Using the same WM TEM images and DG counting criteria, agreements among different technologists (n=5) improved from 60% to 95%. Finally, DGC reference range study using 128 healthy donor samples (age range 18, 72 years) and scoring 100-200 platelets from each donor showed that the DGC distribution of each donor sample was left-skewed with a median of 15% of empty platelets (range: 3, 40). The median DGC was 2.6 with a range of 1.0—5.1. Reference ranges established at the lower 95th percentile for DGC empty platelets is ≤ 31% (95% CI 28, 34) and for the mid-95th percentiles of DGCs between 1.5 and 4.0. DGCs were weakly associated with MPV and IPF (ρ between 0.22 and 0.28); and inversely associated with the percentage of empty platelets (ρ between -0.26 and -0.25). DGC showed no significant association with PC, platelet maximum aggregation or primary slope of aggregation, nor age or gender. Conclusion We validated platelet WM TEM procedures and for the first time established an adult reference range for platelet DGC. The results of association analyses implied that immature platelets may have slightly more DGs. Disclosures: No relevant conflicts of interest to declare.

Platelet-arterial wall interaction appears to be important in the genesis of atherosclerosis. Pigs with homozygous von Willebrand’s disease (vWd) appear resistant to atherosclerosis. We investigated whether there is impairment in platelet-arterial wall interaction in porcine vWd. Superficial endothelial injury was produced in a 4 cm segment of carotid artery by drying for 3 minutes in a stream of air which entered one end (100 ml/min) and exited through a small hole in the other end. This procedure was performed in 9 normal and 6 vWd pigs 24 hours after the I.V. infusion of III-Indium-labeled platelets. Carotid blood flow was re-established and the pigs were sacrificed 24 hours later and the carotids fixed in glutaraldehyde.The ratio of radioactivity (cpm/g) of injured/noninjured carotid arteries was markedly decreased in the vWd pigs. In addition, transmission and scanning electron microscopy confirmed superficial endothelial damage in all injured segments, but only significant platelet deposition in the normal pig injured segments.After a mild endothelial injury, this “in vivo” evidence of impaired platelet-arterial wall interaction in vWd may be related to the resistance to atherosclerosis in these animals.

Abstract Performance on specialized diagnostic tests for platelet disorders, including dense-granule deficiency, is rarely evaluated by external quality assessment (EQA). Members of the North American Specialized Coagulation Laboratory Association that evaluate platelet dense-granule deficiency commonly use whole-mount electron microscopy (EM) methods. This observation led us to develop a pilot EQA survey with standardized EM images and clinical samples on grids from a healthy control subject and a subject with dense-granule deficiency. The survey participants were 8 centers, including 2 with no experience in platelet whole mount EM. All participants, including inexperienced sites, correctly interpreted findings for the normal and dense-granule-deficient platelets. Among experienced sites, agreement was excellent (>82%) on platelet structures to count or not count as dense granules. Participants indicated that future EQA challenges should include clinical samples on grids and standardized images. This is the first report that platelet EM can be assessed by EQA.