Diagnostic laboratory standardization and validation of platelet transmission electron microscopy.

Platelet transmission electron microscopy (PTEM) is considered the gold standard test for assessing distinct ultrastructural abnormalities in inherited platelet disorders (IPDs). Nevertheless, PTEM remains mainly a research tool due to the lack of standardized procedures, a validated dense granule (DG) count reference range, and standardized image interpretation criteria. The aim of this study was to standardize and validate PTEM as a clinical laboratory test. Based on previously established methods, we optimized and standardized preanalytical, analytical, and postanalytical procedures for both whole mount (WM) and thin section (TS) PTEM. Mean number of DG/platelet (plt), percentage of plts without DG, platelet count (PC), mean platelet volume (MPV), immature platelet fraction (IPF), and plt light transmission aggregometry analyses were measured on blood samples from 113 healthy donors. Quantile regression was used to estimate the reference range for DG/plt, and linear regression was used to assess the association of DG/plt with other plt measurements. All PTEM procedures were standardized using commercially available materials and reagents. DG interpretation criteria were established based on previous publications and expert consensus, and resulted in improved operator agreement. Mean DG/plt was stable for 2 days after blood sample collection. The median within patient coefficient of variation for mean DG/plt was 22.2%; the mean DG/plt reference range (mid-95th %) was 1.2-4.0. Mean DG/plt was associated with IPF (p = .01, R2 = 0.06) but not age, sex, PC, MPV, or plt maximum aggregation or primary slope of aggregation (p > .17, R2 < 0.02). Baseline ultrastructural features were established for TS-PTEM. PTEM was validated using samples from patients with previously established diagnoses of IPDs. Standardization and validation of PTEM procedures and interpretation, and establishment of the normal mean DG/plt reference range and PTEM baseline ultrastructural features, will facilitate implementation of PTEM as a valid clinical laboratory test for evaluating ultrastructural abnormalities in IPDs.

Comprehensive Platelet Phenotypic Laboratory Testing and Bleeding History Scoring for Diagnosis of Suspected Hereditary Platelet Disorders: A Single-Institution Experience.

OBJECTIVES: Patients with hereditary/congenital platelet disorders (HPDs) have a broad range of clinical manifestations and laboratory phenotypes. We assessed the performance characteristics of the International Society on Thrombosis and Haemostasis bleeding assessment tool (ISTH-BAT) and clinically validated platelet laboratory tests for diagnosis of HPDs. METHODS: The records of 61 patients with suspected HPDs were reviewed and ISTH-BAT scores calculated. RESULTS: Nineteen (31%) patients had thrombocytopenia, and 46 (75%) had positive ISTH-BAT scores. Thirteen and 17 patients had prolonged PFA-100 (Dade Behring, Miami, FL) adenosine diphosphate and epinephrine closure times, respectively. Twenty-two had abnormal platelet light transmission aggregation. Twenty-four had platelet transmission electron microscopy (PTEM) abnormalities (10 dense granule deficiency, 14 other ultrastructural abnormalities). Positive ISTH-BAT scores were associated with thrombocytopenia (P < .0001) and abnormal PTEM (P = .002). Twenty-three patients had normal results. CONCLUSIONS: ISTH-BAT identified patients with suspected HPDs but lacked a robust association with laboratory abnormalities. Despite comprehensive laboratory testing, some patients may have normal results.

Practice patterns in the diagnosis of inherited platelet disorders within a single institution.

: The diagnosis of inherited platelet disorders (IPDs) is challenging with variable diagnostic practices existing between institutions. To determine patterns and utility of diagnostic testing practices for IPDs within a single institution, a retrospective cohort study was performed. Records of 50 patients (50% women), median age 32 years (1 day to 81 years) were analyzed. In total, 28 (53%) had a positive International Society of Thrombosis and Hemostasis Bleeding Assessment Tool score. Test-ordering patterns were highly variable. All patients had platelet morphology analysis by light microscopy. In total, 42 (84%) underwent light transmission aggregometry, 43 (86%) platelet function analyzer, 37 (74%) platelet electron microscopy, 25 (50%) flow cytometry, and 15 (30%) genetic testing. Platelet function analyzer and light transmission aggregometry were always used as first-order tests, followed by platelet transmission electron microscopy and flow cytometry (81 and 84%, respectively). Genetic testing was obtained up front in five cases (33% of orders), mostly in patients with syndromic thrombocytopenia or in the setting of a known genetic disorder. Test-ordering practices did not adhere to published algorithms. Even within a single institution, great heterogeneity exists in the testing approach to IPDs. Although, a large proportion of cases were studied with platelet transmission electron microscopy and flow cytometry, standard platelet assays established the diagnosis in a great majority. Standardization of testing practices, first beginning at the institutional level is a much needed step forward.

Reactive oxygen species and human platelet GP IIb/IIIa receptor activation.

This study concerned reactive oxygen species for their potential to activate human platelet GP IIb/IIIa receptors. All cells produce reactive oxygen species - radicals that can abstract electrons and hydrogen atoms from biological molecules to alter cell function. In many cells, radicals contribute to cellular signaling. In platelets, the predominant oxidant effect is platelet activation. Less is known concerning oxidants and GP IIb/IIIa receptor activation. The first aim of the current study was to confirm that although both H(2)O(2) and tert butyl hydroperoxide both predispose platelets to aggregation; neither directly activates GP IIb/IIIa receptors. The second aim was to demonstrate that even in the presence of extracellular redox iron; H(2)O(2) does not activate GP IIb/IIIa receptors. The third aim was to determine if extracellular superoxide anions evoke GP IIb/IIIa activation. Finally, a role for intra-platelet iron in GP IIb/IIIa activation was examined. Intracellular superoxide anions are produced in excess during platelet activation and curiously, they are uniquely able to increase intracellular free iron. This iron can, in a redox manner, generate radicals and these iron dependent species modulate signaling systems, including systems associated with adhesion receptor activation. In the current studies, platelets in suspension were exposed to H(2)O(2) and to tert butyl hydroperoxide, to H(2)O(2) plus ferrous or ferric chloride (+/- ascorbate to enhance iron redox cycling) and to xanthine plus xanthine oxidase to generate extra-platelet superoxide anions. Intra-platelet iron was increased with iron ionophore 8-hydroxyquinoline. During flow cytometry, intra-platelet oxidant state was assessed with the redox sensitive fluorescent indicator H2DCF, while GP IIb/IIIa activation was assessed using fluorescent antibody PAC-1. Results showed that although all the oxidizing systems examined increased intra-platelet oxidant state, GP IIb/IIIa receptors were not activated by H(2)O(2), by tert butyl hydroperoxide, by H(2)O(2) plus iron (+/- ascorbate) or by xanthine plus xanthine oxidase. In contrast, iron plus ionophore 8-hydroxyquinoline evoked GP IIb/IIIa activation. Platelet positivity for PAC-1 increased from 2 +/- 0.2 to 28 +/- 7% (P < 0.005). However this response, although vigorous, was less than 56 +/- 8% (P < 0.001) evoked by thrombin 0.1 milliunit/ml. In conclusion, the results indicated that oxidant systems external to platelets did not activate GP IIb/IIIa receptors while increased intra-platelet iron was associated with appearance of cytosolic oxidizing species and with GP IIb/IIIa receptor activation.

A phase II study of ABT-510 (thrombospondin-1 analog) for the treatment of metastatic melanoma.

OBJECTIVES: Thrombospondins are natural inhibitors of angiogenesis, tumor metastases, and tumor growth (melanoma). ABT-510 is a synthetic analog of thrombospondin-1, well tolerated in phase I studies. We conducted a phase II trial evaluating the clinical efficacy of ABT-510 and its effects on biomarkers of angiogenesis and immunity in patients with metastatic melanoma (MM). PATIENTS AND METHODS: A 2-stage phase II clinical trial was conducted to assess the clinical efficacy, safety, and pharmacodynamic effects (angiogenesis and immunity) of ABT-510 in patients with stage IV melanoma. The primary endpoint was 18-week treatment failure rate. Patients self-administered 100 mg of ABT-510 subcutaneously twice daily. Blood samples were collected at baseline and every 3 weeks while on therapy. Eligible patients demonstrated measurable disease, good performance status and no evidence of intracranial metastases. Correlative laboratory studies evaluated biomarkers of angiogenesis and immunity. RESULTS: Twenty-one patients were enrolled. Most patients were stage M1c (71%) and all had prior therapy for MM. Only 3 of the first 20 patients enrolled were progression free and on treatment at 18 weeks resulting in early termination of the study. Decreases in peripheral blood VEGF-A levels and VEGF-C levels, and CD146 and CD34/133 counts relative to pretreatment were detected. Limited changes in antitumor T cell immunity were observed. CONCLUSIONS: ABT-510 therapy administered at 100 mg twice/day in patients with MM did not demonstrate definite clinical efficacy. Further dose escalation or combination with cytotoxic therapy may be more effective therapeutically.

Mitochondrial injury and caspase activation by the local anesthetic lidocaine.

BACKGROUND: Lidocaine, a local anesthetic, can be neurotoxic. However, the cellular mechanisms of its neurotoxicity at concentrations encountered during spinal anesthesia remain unclear. METHODS: The authors examined the mechanisms of lidocaine neurotoxicity in the ND7 cell line derived from rat dorsal root ganglion. Individual neurons were assayed by flow cytometry or microscopy using fluorescent probes of plasma membrane integrity, mitochondrial membrane potential, caspase activity, phospholipid membrane asymmetry, and mitochondrial cytochrome c release. RESULTS: In the ND7 cell line, lidocaine at 185 mm x 10 min to 2.3 mm x 24 h caused necrosis or late apoptosis. Equimolar Tris buffer and equipotent tetrodotoxin controls were not toxic, indicating that neither osmotic nor Na-blocking effects explain lidocaine neurotoxicity. The earliest manifestation of lidocaine neurotoxicity was complete loss of mitochondrial membrane potential within 5 min after exposure to lidocaine at a concentration of 19 mm or greater. Consistent with these data, 37 mm lidocaine (1%) induced release of mitochondrial cytochrome c into the cytoplasm, as well as plasma membrane blebbing, loss of phosphatidylserine membrane asymmetry, and caspase activation, with release of mitochondrial cytochrome c to the cytoplasm within 2 h. Treatment with z-VAD-fmk, a specific inhibitor of caspases, prevented caspase activation and delayed but did not prevent neuronal death, but did not inhibit the other indicators of apoptosis. CONCLUSIONS: Collectively, these data indicate that lidocaine neurotoxicity involves mitochondrial dysfunction with activation of apoptotic pathways.

Effect of local anesthetic on neuronal cytoplasmic calcium and plasma membrane lysis (necrosis) in a cell culture model.

BACKGROUND: To investigate the mechanism by which rare cases of spinal local anesthetic (LA) neurotoxicity occur, we have tested the hypotheses that LAs elevate cytoplasmic calcium (Ca2+(cyt)), that this is associated with a neurotoxic effect, and that lidocaine and bupivacaine differ in their neurotoxicity. METHODS: Neurons of the ND7 cell culture line, derived from dorsal root ganglion, were loaded with fura-2 and analyzed by digitized video fluorescence microscopy during 60 min LA exposure, allowing determination of Ca2+(cyt) and time of necrotic cell death (plasma membrane lysis) at the single neuron level. RESULTS: Lidocaine 0.1% and bupivacaine 0.025% caused minimal changes in Ca. Lidocaine 0.5-5% and bupivacaine 0.125-0.625% caused an early, small (less than threefold), concentration-dependent increase in Ca2+(cyt) that was transient and returned to near baseline within 10 min. Lidocaine 2.5% and 5% then caused a sustained, greater than ten-fold increase in Ca2+(cyt) and death in some neurons during the 60 min exposure period. Pretreatment with thapsigargin eliminated the initial transient increase in Ca2+(cyt), consistent with endoplasmic reticulum (ER) as its source, and increased neuronal death with 5% lidocaine, suggesting that lidocaine neurotoxicity can be increased by failure of ER to take up elevated Ca2+(cyt). The later sustained increase in Ca2+(cyt) seen with 2.5 and 5% lidocaine was prevented in Ca2+ -free medium, and restored when Ca2+ was added back to the buffer in the presence of lidocaine, suggesting that higher concentrations of lidocaine increase influx of Ca2+ through the plasma membrane. CONCLUSIONS: In this model, lidocaine greater than 2.5% elevates Ca2+(cyt) to toxic levels. Bupivacaine and lower concentrations of lidocaine transiently alter Ca2+(cyt) homeostasis for several minutes, but without an immediate neurotoxic effect within 60 min.