Mean platelet diameter measurements to classify inherited thrombocytopenias.

INTRODUCTION: Mean platelet volume (MPV) assists the differential diagnosis of inherited thrombocytopenia (IT) but lacks standardisation and varies between automated analysers. Classification of IT based on mean platelet diameter (MPD) has been proposed by an international collaborative study but has not been validated. METHODS: To assess the applicability of MPD to classify forms of IT, digital images of blood films from patients with established genetic causes for IT were generated, and the MPD measured (ZEISS Axio-scanner and Image J software) by a blinded reviewer. Comparison was made to the proposed classification system. RESULTS: Mean platelet volume was measured in thrombocytopenia with different genetic aetiologies, bilallelic BSS (bBSS) (n = 1), monoallelic BSS (mBSS) (n = 2), MYH9-related disorders (MYH9-RD) (n = 11), GFI1B-related thrombocytopenia (RT) (n = 15), FLI1-RT (n = 2), TUBB1-RT (n = 3), ITGA2B/ITGB3-RT (n = 1), RUNX1-RT (n = 2) and controls (n = 54). bBSS and 82% of MYH9-RD samples had MPD >4 µm which correlated with "IT with giant platelets." Only 55% of samples expected in the "large platelet group" had MPD meeting the classification cut-off (MPD >3.2 µm). FLI1-RT MPD were significantly larger than expected whilst ITGA2B/ITGB3-RT MPD were smaller than proposed. MPD in FPD/AML were "normal." CONCLUSION: Platelet MPD measurements are a useful guide to classify IT, but the time taken to record measurements may limit clinical applicability.

Receptor homodimerization plays a critical role in a novel dominant negative P2RY12 variant identified in a family with severe bleeding.

Essentials Three dominant variants for the autosomal recessive bleeding disorder type-8 have been described. To date, there has been no phenotype/genotype correlation explaining their dominant transmission. Proline plays an important role in P2Y12R ligand binding and signaling defects. P2Y12R homodimer formation is critical for the receptor function and signaling. SUMMARY: Background Although inherited platelet disorders are still underdiagnosed worldwide, advances in molecular techniques are improving disease diagnosis and patient management. Objective To identify and characterize the mechanism underlying the bleeding phenotype in a Caucasian family with an autosomal dominant P2RY12 variant. Methods Full blood counts, platelet aggregometry, flow cytometry and western blotting were performed before next-generation sequencing (NGS). Detailed molecular analysis of the identified variant of the P2Y12 receptor (P2Y12R) was subsequently performed in mammalian cells overexpressing receptor constructs. Results All three referred individuals had markedly impaired ADP-induced platelet aggregation with primary wave only, despite normal total and surface P2Y12R expression. By NGS, a single P2RY12:c.G794C substitution (p.R265P) was identified in all affected individuals, and this was confirmed by Sanger sequencing. Mammalian cell experiments with the R265P-P2Y12R variant showed normal receptor surface expression versus wild-type (WT) P2Y12R. Agonist-stimulated R265P-P2Y12R function (both signaling and surface receptor loss) was reduced versus WT P2Y12R. Critically, R265P-P2Y12R acted in a dominant negative manner, with agonist-stimulated WT P2Y12R activity being reduced by variant coexpression, suggesting dramatic loss of WT homodimers. Importantly, platelet P2RY12 cDNA cloning and sequencing in two affected individuals also revealed three-fold mutant mRNA overexpression, decreasing even further the likelihood of WT homodimer formation. R265 located within extracellular loop 3 (EL3) is one of four residues that are important for receptor functional integrity, maintaining the binding pocket conformation and allowing rotation following ligand binding. Conclusion This novel dominant negative variant confirms the important role of R265 in EL3 in the functional integrity of P2Y12R, and suggests that pathologic heterodimer formation may underlie this family bleeding phenotype.

Thrombocytopenia and CD34 expression is decoupled from α-granule deficiency with mutation of the first growth factor-independent 1B zinc finger.

Essentials The phenotypes of different growth factor-independent 1B (GFI1B) variants are not established. GFI1B variants produce heterogeneous clinical phenotypes dependent on the site of mutation. Mutation of the first non-DNA-binding zinc-finger causes a mild platelet and clinical phenotype. GFI1B regulates the CD34 promoter; platelet CD34 expression is an indicator of GFI1B mutation. SUMMARY: Background Mutation of the growth factor-independent 1B (GFI1B) fifth DNA-binding zinc-finger domain causes macrothrombocytopenia and α-granule deficiency leading to clinical bleeding. The phenotypes associated with GFI1B variants disrupting non-DNA-binding zinc-fingers remain uncharacterized. Objectives To determine the functional and phenotypic consequences of GFI1B variants disrupting non-DNA-binding zinc-finger domains. Methods The GFI1B C168F variant and a novel GFI1B c.2520 + 1_2520 + 8delGTGGGCAC splice variant were identified in four unrelated families. Phenotypic features, DNA-binding properties and transcriptional effects were determined and compared with those in individuals with a GFI1B H294 fs mutation of the fifth DNA-binding zinc-finger. Patient-specific induced pluripotent stem cell (iPSC)-derived megakaryocytes were generated to facilitate disease modeling. Results The DNA-binding GFI1B variant C168F, which is predicted to disrupt the first non-DNA-binding zinc-finger domain, is associated with macrothrombocytopenia without α-granule deficiency or bleeding symptoms. A GFI1B splice variant, c.2520 + 1_2520 + 8delGTGGGCAC, which generates a short GFI1B isoform that lacks non-DNA-binding zinc-fingers 1 and 2, is associated with increased platelet CD34 expression only, without quantitative or morphologic platelet abnormalities. GFI1B represses the CD34 promoter, and this repression is attenuated by different GFI1B zinc-finger mutations, suggesting that deregulation of CD34 expression occurs at a direct transcriptional level. Patient-specific iPSC-derived megakaryocytes phenocopy these observations. Conclusions Disruption of GFI1B non-DNA-binding zinc-finger 1 is associated with mild to moderate thrombocytopenia without α-granule deficiency or bleeding symptomatology, indicating that the site of GFI1B mutation has important phenotypic implications. Platelet CD34 expression appears to be a common feature of perturbed GFI1B function, and may have diagnostic utility.

In situ diagnostic methods for catheter related bloodstream infection in burns patients: A pilot study.

BACKGROUND: One of the most common and potentially fatal complications in critically ill burns patients is catheter related bloodstream infection (CR-BSI). Lack of in situ diagnostic techniques requires device removal if CR-BSI is suspected with 75-85% of catheters withdrawn unnecessarily. AIMS: To assess the sensitivity, specificity and accuracy of two in situ diagnostic methods for CR-BSI in an adult ICU burns population: Differential Time to Positivity (DTP) and Semi-Quantitative Superficial Cultures (SQSC). METHODS: Both arterial (AC) and central venous (CVC) catheters were studied. On clinicians' suspicion of CR-BSI, the CVC and AC were removed. Superficial semi-quantitative cultures were taken by removing the dressings and swabbing within a 3cm radius of the CVC and AC insertion sites, as well as inside each hub of the CVC and AC. Peripheral blood was taken for qualitative culture and the catheter tip sent for semi-quantitative culture. DTP was considered positive if culture of lumen blood became positive at least 120min before peripheral blood with an identical pathogen. Superficial and tip cultures were identified as positive if ≥15 CFUs were grown. CR-BSI was confirmed when both catheter tip culture and peripheral blood culture were positive with the same micro-organism. RESULTS: Sixteen patients (88% male) with an APACHE II score of 22.0 (7.3) were enrolled. The mean age was 45.7 (16.9) years with mean total burn surface area 32.9 (19.4)%. Fifty percent had airway burns. ICU stay was 19.9 (11.1) days. All 16 survived ICU discharge with a hospital survival of 93%. There were 20 episodes of CR-BSI in these 16 patients. For these 20 episodes the exposure time (line days) was 113.15. The CR-BSI rate was 15.6 per 1000 catheter days (95% CI 1.9-56.4). For diagnosis of CR-BSI in either AC and CVC, SQSC had a sensitivity of 50% [95% CI 3-97], specificity 83.3% [95% CI 67-93], PPV 14.3 [95% CI 1-58], NPV 96.8 [95% CI 81-100], accuracy of 81.6% [95%CI 65-92] and diagnostic odds ratio 5.0 [95% CI 0.3-91.5]. To diagnose tip colonisation (>15CFU), sensitivity of SQSC was 75% [95% CI 22-99], specificity 88.2% [95%CI 72-96], PPV 42.7 [95% CI 12-80], NPV96.8% [95% CI 81-100], accuracy 86.8% [95% CI 71-95] and diagnostic odds ratio 22.5 [95% CI 1.9-271.9]. For combined DTP blood cultures, sensitivity for CR-BSI was 50% [95% CI 3-97], with specificity 97% [95% CI 82-100], PPV 50% [5% CI 3-97%], NPV 97% [95% CI 82-100], accuracy 94.3% 95% CI 79-99] and diagnostic odds ratio 32 [95% CI 1.1-970.8]. CONCLUSION: Both DTP and SQSC displayed high specificity, NPV and accuracy in a population of adult burns patients. These features may make these tests useful for ruling out CR-BSI in this patient group. This study was limited by a low number of events and further research is required.

Multiple squamous cell carcinomas following introduction of nilotinib.

BACKGROUND: Nilotinib is a second generation tyrosine kinase inhibitor, used in the treatment of chronic myelogenous leukaemia (CML). METHODS: We assessed a 72-year-old woman who was treated with nilotinib for CML. RESULTS: During the year following commencement of nilotinib, the patient developed eight squamous cell carcinomas (SCCs) on her legs. CONCLUSIONS: Development of SCC is a previously unreported adverse reaction to nilotinib.

Systemic capillary leak syndrome: recognition prevents morbidity and mortality.

Idiopathic systemic capillary leak syndrome (SCLS) is extremely rare but carries a high morbidity and mortality. The diagnosis is made clinically by a classic triad of hypotension, hypoalbuminaemia and haemoconcentration. There have been recent advances in understanding the pathophysiological basis for SCLS and in effective prophylaxis. We report a case of SCLS to increase awareness of the condition and to highlight the benefits of prophylactic intravenous immunoglobulin in this condition.