Lingineni, Ravi K.

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.

Background: Understanding the effect size, variability, and underlying physiology of the dawn phenomenon is important for next-generation closed-loop control algorithms for type 1 diabetes (T1D). Subjects and Methods: We used an iterative protocol design to study 16 subjects with T1D on individualized insulin pump therapy for two successive nights. Endogenous glucose production (EGP) rates at 3 a.m. and 7 a.m. were measured with [6,6- 2 H 2 ]glucose as a single tracer, infused from midnight to 7 a.m. in all subjects. To explore possibility of tracer recycling due to prolonged [6,6- 2 H 2 ]glucose infusion, which was highly probable after preplanned interim data analyses, we infused a second tracer, [6- 3 H]glucose, from 4 a.m. to 7 a.m. in the last seven subjects to measure EGP at 7 a.m. Results: Cortisol concentrations increased during both nights, but changes in glucagon and insulin concentration were inconsistent. Although the plasma glucose concentrations rose from midnight to 7 a.m. during both nights, EGP measured with [6,6- 2 H 2 ]glucose between 3 a.m. and 7 a.m. did not differ during Night 1 but fell in Night 2. However, EGP measured with [6- 3 H]glucose at 7 a.m. was higher than that measured with [6,6- 2 H 2 ]glucose during both nights, thereby suggesting tracer recycling probably underestimating EGP calculated at 7 a.m. with [6,6- 2 H 2 ]glucose. Likewise, EGP was higher at 7 a.m. with [6- 3 H]glucose than at 3 a.m. with [6,6- 2 H 2 ]glucose during both nights. Conclusions: The data demonstrate a consistent overnight rise in glucose concentrations through increased EGP, mediated likely by rising cortisol concentrations. The observations with the dual tracer approach imply significant tracer recycling leading to underestimation of EGP measured by longer-duration tracer infusion.

OBJECT Tobacco smoking is one of the most important risk factors for the formation of intracranial aneurysms and for aneurysmal subarachnoid hemorrhages. Smoking has also been suggested to contribute to the recurrence of aneurysms after endovascular coiling. To improve the understanding of the impact of smoking on long-term outcomes after coil embolization of intracranial aneurysms, the authors studied a consecutive contemporary series of patients treated at their institution. The aims of this study were to determine whether smoking is an independent risk factor for aneurysm recurrence and retreatment after endovascular coiling. METHODS All patients who had received an intrasaccular coil embolization of an intracranial aneurysm, who had undergone a follow-up imaging exam at least 6 months later, and whose smoking history had been recorded from January 2005 through December 2012 were included in this study. Patients were stratified according to smoking status into 3 groups: 1) never a smoker, 2) current smoker (smoked at the time of treatment), and 3) former smoker (quit smoking before treatment). The 2 primary outcomes studied were aneurysm recurrence and aneurysm retreatment after treatment for endovascular aneurysms. Kruskal-Wallis and chi-square tests were used to test statistical significance of differences in the rates of aneurysm recurrence, retreatment, or of both among the 3 groups. A multivariate logistic regression analysis controlling for smoking status and for several characteristics of the aneurysm was also performed. RESULTS In total, 384 patients with a combined total of 411 aneurysms were included in this study. The aneurysm recurrence rate was not significantly associated with smoking: both former smokers (OR 1.00, 95% CI 0.61–1.65; p = 0.99) and current smokers (OR 0.58, 95% CI 0.31–1.09; p = 0.09) had odds of recurrence that were similar to those who were never smokers. Former smokers (OR 0.78, 95% CI 0.46–1.35; p = 0.38) had odds of retreatment similar to those of never smokers, and current smokers had a lower odds of undergoing retreatment (OR 0.44, 95% CI 0.21–0.91; p = 0.03) than never smokers. Moreover, an analysis adjusting for aneurysm rupture, diameter, and initial occlusion showed that former smokers (OR 0.65, 95% CI 0.33–1.28; p = 0.21) and current smokers (OR 1.04, 95% CI 0.60–1.81; p = 0.88) had odds of aneurysm recurrence similar to those who were never smokers. Adjusting the analysis for aneurysm rupture, diameter, and occlusion showed that both former smokers (OR 0.49, 95% CI 0.23–1.05; p = 0.07) and current smokers (OR 0.82, 95% CI 0.46–1.46; p = 0.50) had odds of retreatment similar to those of patients who were never smokers. CONCLUSIONS The results show that smoking was not an independent risk factor for aneurysm recurrence and aneurysm retreatment among patients receiving endovascular treatment for intracranial aneurysms at the authors' institution. Nonetheless, patients with intracranial aneurysms should continue to be counseled about the risks of tobacco smoking.

Abstract BACKGROUND Anticoagulation protocols used during mechanical circulatory support call for titration of antiplatelet agents. We compared the precision and reliability of 5 platelet function tests in healthy volunteers and donors on daily antiplatelet therapy to distinguish their efficacy for titrating antiplatelet therapy. METHODS We assessed arachidonic acid–induced platelet function by light transmission aggregometry (LTA), Multiplate impedance aggregometry, VerifyNow, and platelet mapping by thromboelastography (TEG PM). We assessed ADP-induced platelet function by the same methods and flow cytometry. Forty healthy volunteers and 10–13 volunteers on daily aspirin and/or clopidogrel therapy were evaluated. We compared tests for intraassay precision, interassay precision (samples from 2 separate blood draws), and reliability coefficient. RESULTS For arachidonic acid–induced platelet aggregation in healthy volunteers, intra- and interassay CVs were ≤10% for all methods. Intra- and interassay precision among donors on daily aspirin was ≤30% for all methods except LTA (38% interassay CV) and TEG PM (95% intraassay and 104% interassay CV). For ADP-induced platelet function, intra- and interassay precision was ≤10% and ≤30% for all methods. Only Multiplate demonstrated moderate or greater (R > 0.40) reliability coefficients for arachidonic acid–induced platelet function among all subjects. All methods of ADP-induced platelet function, except TEG PM, demonstrated substantial or greater (R > 0.60) reliability among all subjects. CONCLUSIONS TEG PM is least suited to monitor effects of antiplatelet agents. Multiplate impedance aggregometry was the only method to demonstrate an acceptable reliability coefficient among healthy volunteers and donors on both aspirin and clopidogrel therapy.

BackgroundThe use of hematopoietic progenitor cell (HPC) transplant has risen over the past two decades. A variety of adverse events (AEs) of varying severity have been noted during HPC infusions. These AEs have been associated with several factors such as the amount of dimethyl sulfoxide and white blood cells in the HPC product. We performed a single‐institution retrospective analysis to determine the effect of two different HPC infusion techniques, manual push with syringes versus infusion from bags with the aid of gravity, on the occurrence of infusion‐related AEs.Study Design and MethodsInfusions between December 2008 and November 2010 involving peripheral blood HPCs were reviewed. Pertinent clinical and HPC product–related information was recorded. Data were analyzed to determine the incidence of infusion‐related AEs and its association with patient and product‐related variables.ResultsWe found 461 AEs in 645 patients during the study period. A total of 325 (50%) experienced at least one AE. Flushing was the most common type of AE followed by nausea and hypertension. The use of syringe infusion was more commonly associated with AEs (odds ratio, 1.82 [95% confidence interval, 1.32‐2.50]; p = 0.002). Other independent risk factors were cryopreserved products and the amount of polymorphonuclear leukocytes in the product.ConclusionTo our knowledge, this is the first study examining the effect of two different infusion techniques on infusion‐related AEs. Our findings suggest that the use of bags for infusion protected the patients from AEs.

Background: Currently, patients with type 1 diabetes decide on the amount of insulin to administer based on several factors, including current plasma glucose value, expected meal input, and physical activity (PA). One future therapeutic modality for patients with type 1 diabetes is the artificial endocrine pancreas (AEP). Incorporation of PA could enhance the efficacy of AEP significantly. We compared the main technologies used for PA quantitation. Subjects and Methods: Data were collected during inpatient studies involving healthy control subjects and type 1 diabetes. We report PA quantified from accelerometers (acceleration units [AU]) and heart rate (HR) monitors during a standardized activity protocol performed after a dinner meal at 7 p.m. from nine control subjects (four were males, 37.4±12.7 years old, body mass index of 24.8±3.8 kg/m 2 , and fasting plasma glucose of 4.71±0.63 mmol/L) and eight with type 1 diabetes (six were males, 45.2±13.4 years old, body mass index of 25.1±2.9 kg/m 2 , and fasting plasma glucose of 8.44±2.31 mmol/L). Results: The patient-to-patient variability was considerably less when examining AU compared with HR monitors. Furthermore, the exercise bouts and rest periods were more evident from the data streams when AUs were used to quantify activity. Unlike the AU, the HR measurements provided little insight for active and rest stages, and HR data required patient-specific standardizations to discern any meaningful pattern in the data. Conclusions: Our results indicated that AU provides a reliable signal in response to PA, including low-intensity activity. Correlation of this signal with continuous glucose monitoring data would be the next step before exploring inclusion as input for AEP control.

BACKGROUNDPatients diagnosed with primary sclerosing cholangitis (PSC) and dominant strictures often undergo endoscopic retrograde cholangiopancreatography with brush cytology to exclude or confirm the development of malignancy. Equivocal (atypical or suspicious) routine cytologic results may confound patient management decisions, especially in the absence of a mass on imaging. The objective of the current study was to identify independent predictors of malignancy in patients with PSC with an equivocal cytology diagnosis.METHODSPatients with PSC underwent brush cytology for routine cytology and fluorescence in situ hybridization (FISH) during endoscopy as per standard care. FISH slides were classified as polysomy if at least 5 cells displayed a gain of ≥ 2 probes. A retrospective search identified 102 patients without a mass lesion noted on initial imaging studies, an equivocal routine cytology, and ≥ 2 years of follow‐up.RESULTSOf 102 patients, 30 (29%) with an equivocal cytology result developed cancer within 2 years. Serum carbohydrate antigen 19‐9 (CA 19‐9) levels ≥ 129 U/mL (hazard ratio [HR] 3.19; P = .001) and polysomy (HR 8.70; P < .001) were each found to be predictive of cancer. Of 10 patients who had elevated CA 19‐9 levels and polysomy, all went on to develop cancer (9 within 2 years). Although only 10 patients were included in this subset, the combination of elevated CA 19‐9 and polysomy was found to be predictive of cancer (HR 10.92; P < .001).CONCLUSIONSPolysomy by FISH identified those patients most likely to have or develop malignancy in the challenging clinical scenario of PSC with no mass at baseline and equivocal cytology. The combination of an elevated serum CA 19‐9 level with polysomy is highly suspicious for the presence of malignancy. Cancer (Cancer Cytopathol) 2013;121:708–717. © 2013 American Cancer Society.