Standard Morphological Criteria Research Articles

Algorithms for the analysis of polysomnographic recordings with customizable criteria

The diagnosis of Sleep Apnoea–Hypopnoea Syndrome requires the visual inspection of a recording containing a large number of physiological parameters of the patient – the polysomnogram. The purpose of this visual inspection is the identification and characterization of different types of pathological events that occur over these parameters. These events are defined by a set of morphological criteria. Based on these criteria, commercial tools have been developed to support clinicians in the task of visually inspecting polysomnograms. This article argues that the standard morphological criteria are just guiding recommendations that experienced physicians often adapt to each specific diagnostic context. Thus, tools that support the analysis of polysomnograms ideally should use flexible criteria that could be easily customizable by clinicians. In this paper, we propose algorithms capable of identifying pathological events relevant in the diagnosis of SAHS using custom criteria that are acquired directly from the clinician. These algorithms take advantage of the Fuzzy Set Theory to capture and handle the vagueness and uncertainty that are characteristics of medical knowledge. Knowledge acquisition using the traditional linguistic approach of the Fuzzy Sets Theory is supported by a desktop tool. However, the authors feel that some of the criteria that need to be acquired are more visual in nature than linguistic. An alternative mechanism for the visual acquisition of these criteria is proposed. Finally, when presenting the pathological events that have been identified, the tool uses several visual metaphors designed to simplify visual inspection of the polysomnogram. We have validated our proposal over 69 h of polysomnographic recordings arising from 12 patients that were subjected to a sleep study. 95.7% of the events identified were correct detections. The rate of false negatives was 1.6%.

Lenalidomide Enhances Clonogenic Activity, Proliferation and Erythroid Lineage Commitment of CD34+ Progenitor Cells While It Is Cytotoxic to CD34- Accessory Cells.

AbstractAbstract 1184Lenalidomide, a second generation immunomodulatory derivative of thalidomide, has demonstrated significant clinical benefit in myelodysplastic syndrome, multiple myeloma and B-cell malignancies. Its side effects include myelosuppression, particularly neutropenia, and impaired filgrastim induced stem cell mobilization in patients with myeloma. The precise mechanism of action of lenalidomide on hematopoietic and bone marrow microenvironment remains largely unknown. In this study, we investigated the effect of lenalidomide on survival, lineage commitment and differentiation of normal purified CD34+ hematopoietic stem cells.Mobilized peripheral blood progenitor cells (PBPCs) were obtained from a normal allogeneic stem cell donor with an informed consent, and were subjected to positive selection with the EasySep magnetic cell selection system to purify CD34+ cells (purity of 95%). Unsorted and CD34- accessory cells were used as controls.To assess the direct cytotoxic and proliferative effect of lenalidomide on purified CD34+ cells, lenalidomide (Celgene, NJ) was dissolved in DMSO and was diluted in culture medium to final concentration of 0.1, 1, 10, or 100 μM. Purified CD34+, CD34- and unsorted cells were plated in triplicates at a density of 2×104/mL in serum-free StemSpan® medium supplemented with growth factors and cytokines. 24 hours after plating the cells, lenalidomide or 0.1% DMSO as control was added. 72 hours following treatment, cells were counted with Beckman coulter counter and phenotypically analyzed using Accuri C6 flow-cytometer. The percentage of viable cells was determined using 7AAD staining. While no difference in viability was noted in the treated vs. untreated purified CD34+ cells, the viability of CD34- cells and unsorted cells was reduced to 30% and 40% respectively by lenalidomide. Contrary to our assumption, lenalidomide, in a dose dependant manner, induced proliferation of purified CD34+ cells. Maximal increase in proliferation was observed between 1mM and 10mM concentrations. The rate of growth plateaued beyond the 10mM concentration and 100mM concentration appeared to have no additional effect. At the peak of growth (10mM), the cell count nearly tripled. (Fig. 1) Lenalidomide appeared to be cytotoxic to CD34- and unsorted cells in a dose dependant manner. At 100mM concentration, the CD34- cell proliferation dropped by 4 fold and the unsorted cells by 7 fold.To examine the effect of lenalidomide on the clonogenic potential of purified CD34+ cells, 2,000 cells were seeded onto MethoCult® media (Stem cell technologies) supplemented with growth factors and cytokines. Cells were cultured in the presence of lenalidomide (0.1, 1, 10 and 100 μM) or 0.1 % DMSO as control. BFU-E, CFU-GM and CFU-mix were scored on day 14 with the use of an inverted microscope and standard morphological criteria. Significance of difference between groups was determined by student's t test for paired samples. Lenalidomide induced a significant increase in clonogenic activity of CD34+ cells (148 ± 27 colonies per 103 CD34+ cells, p = 0.01), most evident at the dose of 10 μM with enhanced formation of BFU-E (102 ± 23/ per 103 CD34+ cells, p = 0.01) but demonstrated no significant impact on CFU-GM (36 ± 14 per 103 CD34+ cells, p = 0.83) compared to control. (Fig. 2)This study demonstrates that lenalidomide has pronounced effects on both CD34+ and CD34- components of mobilized PBPCs. It enhances the proliferative and clonogenic potentials of CD34+ cells with lineage commitment towards erythroid differentiation. It also has cytotoxic activity towards CD34- cells. It is likely that neutropenia and impaired mobilization of stem cells in patients treated with lenalidomide is mediated via its impact on CD34- accessory cells in the bone marrow microenvironment. Further studies exploring the impact of lenalidomide on normal bone marrow stromal cells, T and NK cell mediated signaling are needed. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The Impact of FLT3-ITD Mutation on the Expression of Multidrug Resistance Genes MDR-1, MRP-1, BCRP and LRP In Adult Acute Myeloid Leukemia

AbstractAbstract 1808 BackgroundMultidrug resistance (MDR) is a clinically relevant problem in the treatment of acute myeloid leukaemia (AML). From 20–50% of patients with AML are primarily resistant to induction chemotherapy and in the great majority of those who respond well to initial therapy the disease relapses. While some mechanisms of MDR have been well characterized, this phenomenon still remains incompletely understood. Three ATP-dependent drug transporting proteins: p-glycoprotein (Pgp; MDR-1), multidrug resistant protein-1 (MRP-1), breast cancer resistance protein (BCRP) and one major vault protein-lung resistance-related protein (LRP) and the internal tandem duplication (ITD) in the receptor tyrosine kinase FLT3 have been found to have a strong correlation with MDR and poor outcome in AML. About 30% of AML cases harbor FLT3-ITD mutation. Mainly MDR-1 protein and FLT3-ITD mutation are targets for inhibition by numerous agents in clinical practice. ObjectivesIn this retrospective study we estimated expression of MDR genes encoding MDR-1, MRP-1, BCRP, LRP proteins and detected FLT3-ITD mutation in patients with AML. We divided all patients into two groups according to FLT3-ITD mutation and we compared expression of MDR genes and other prognostic factors in AML: age (<60 vs ≥60 years), cytogenetic/molecular aberrations (good, intermediate, poor), de novo/secondary AML, white blood cell count (WBC), extramedullary involvement, type of AML according to the FAB classification, CD34 expression on blast cells and myeloperoxidase activity (<50% of blast cells, ≥50%) between groups Moreover the rate of complete remissions (CR) and relapses and disease free survival (DFS) and overall survival (OS) in both groups were estimated. Materials and MethodsA total of 126 adult patients at median age of 53 years (range 21–87) with newly diagnosed between June 2005 and March 2010, previously untreated AML and 40 healthy donors were included in this study. Diagnosis of AML was based on standard morphological and immunophenotypical criteria according to the WHO classification. Most of the patients were treated with anthracycline and cytarabine during the induction regime. There were 39 FLT3-ITD positive (30,4%) and 87 FLT3-ITD negative (69,6%) patients. DNA was extracted from bone marrow (90 patients) or peripheral blood (36 patients) samples and used to detect FLT3-ITD with PCR method. RNA was extracted from the same samples and following reverse transcription RQ-PCR method was performed for assessment of expression of MDR-1, MRP-1, BCRP and LRP genes. Parameters of RQ-PCR reaction were established based on the Europe Against Cancer protocol. Every tested sample was amplified simultaneously for the presence of control ABL gene for standardization of mRNA level for tested genes. Results of the expression of tested genes were presented as coefficients calculated with an intermediate method according to Pfaffl's rule. ResultsTo our surprise, significant higher expression of MDR-1 gene was found either in bone marrow or peripheral blood samples of the patients who do not harbor FLT3-ITD in contrast to the patients harboring this mutation (median 0,3025 vs 0,0756, p=0,003 total; median 0,2690 vs 0,0583, p= 0,002 bone marrow samples; median 0,5775 vs 0,0920, p=0,001 peripheral blood samples). For MRP-1, BCRP and LRP genes we did not find significant differences in their expression between two groups. When adjusted for other prognostic factors, only WBC was significantly higher in FLT3-ITD positive group of patients: 1–294×109/l, median 74 vs 0–286×109/l, median 6,5 in FLT3-ITD negative group of patients (p=0,0001). We did not observe significant differences in CR and relapses rates, DFS and OS between groups. ConclusionsIt seems that FLT3-ITD mutation may in some mechanisms influence MDR-1 gene expression We observed significant inhibition of MDR-1 expression in FLT3-ITD positive patients. Because risk factors for AML and outcome of AML therapy did not differ between groups it seems that FLT3-ITD mutation and high expression of MDR-1 gene are independent high risk factors which negatively influence results of AML therapy. These results might provide a powerful risk classification and useful information for treatment strategies including inhibitors of tyrosine kinase or MDR-1 protein.This project was supported by grant of The Ministry of Science (N N402 208935). Disclosures:No relevant conflicts of interest to declare.

Minimizing the diagnosis of “follicular lesion of undetermined significance” and identifying predictive features for neoplasia

We used proposed standard morphologic criteria as a guideline to conduct a 10-year retrospective review of thyroid fine-needle aspiration specimens that were originally interpreted as "follicular lesion of undetermined significance" and followed by surgical intervention. We sought to investigate whether the indeterminate diagnosis could be minimized by assessing various cytomorphologic features and identifying the features predictive of neoplasia. Using the standard morphologic criteria, we semi-quantitatively assessed a total of 24 cytomorphologic features in 123 aspirates and recorded an overall interpretation on completion of the review. Cyto-histologic correlation was evaluated and logistic regression model was performed to identify cytomorphologic features predictive of neoplasia. Although 32 of 123 aspirates remained in the indeterminate category, the retrospective review reclassified 64 aspirates as non-neoplasia and 27 aspirates as neoplasia. Histologic confirmation was achieved in 47 (73.4%) non-neoplastic and 15 (55.6%) neoplastic aspirates with a diagnostic accuracy of 68.1%. Furthermore, our analysis demonstrated that neoplasia is positively associated with the presence of syncytial tissue fragments, isolated microfollicles, follicles with scalloped borders, scant cytoplasm, irregular nuclear membranes, nuclear overlapping, coarse chromatin, and increased cellularity. On the contrary, the presence of honeycombing tissue fragments, background colloid, and histiocytes inversely correlated with neoplasia. Overall, using proposed standard morphological criteria can minimize the diagnosis of "follicular lesion of undetermined significance," and allow for more accurate cyto-histologic correlation, and thereby playing a substantial role in reducing unnecessary surgical intervention.

In vitro maturation (IVM) of murine and human germinal vesicle (GV)–stage oocytes by coculture with immortalized human fallopian tube epithelial cells

To improve the maturation rate of murine and human germinal vesicle (GV) oocytes using human tubal epithelial cells (hTECs). Murine and human GV oocytes were randomized to human tubal fluid (HTF) media alone or cocultured with mouse embryonic fibroblasts (MEFs) or primary hTECs or immortalized hTECS (ihTECs) for various times. Rates of maturation to meiosis II (MII) were compared between groups. TECs were isolated from discarded salpingectomy specimens. One batch was immortalized with TERT and SV40 large T-antigen. GV oocytes (n = 710) were isolated from 8-week-old-mice at 40 hours after pregnant mare's serum gonadotropin stimulation. Discarded human GV oocytes (n = 62) were obtained from intracytoplasmic sperm injection cycle IVF center patients. Oocytes were cultured in HTF media alone or with MEFs, hTECs, or ihTECs. Maturation rates were assessed by standard morphological criteria and compared. The maturation rate of murine GV oocytes to MII at 12 and 24 hours increased significantly in coculture with hTECS and ihTECS compared with MEF and HTF media alone. In addition, the development rate after IVF was significantly higher with hTECS and ihTECS than in MEF and HTF media alone. Maturation of human GV oocytes to MII at 24 and 48 hours was significantly higher in hTECS and ihTECS compared with HTF media alone. Coculture with either primary or immortalized TECs might improve oocyte quality and significantly raise in vitro maturation rates for GV oocytes.

How Fast Can Acute Promyelocytic Leukemia Be Diagnosed? - Evaluation of PML Immunofluorescence, Flowcytometric Immunophenotypic Analysis and RT-PCR for PML/RARa.

Abstract 4674 IntroductionAcute promyelocytic leukemia (APL) constitutes nearly 5-8% of all leukemias, however its frequency is higher in some populations. It is essential to diagnose APL rapidly and accurately as it often presents as a devastating coagulopathy and shows unique sensitivity to all-trans retinoic acid (ATRA). A great deal of morphological, immunophenotypic and cytogenetic heterogeneity of APL adversely affects efforts for providing an accurate and rapid diagnosis. APL is associated with t(15;17)(q22;q12) with generation of a novel PML/RARa fusion protein in 95% cases. Cases positive for t(15;17)/ PML/RARa are ATRA sensitive, while some of those without it [eg t(11;17)(q23:q21)] are not. Several techniques such as karyotyping, fluorescent in situ hybridization (FISH), and reverse –transcriptase polymerase chain reaction (RT-PCR), used for its detection are time consuming, laborious, costly and require specialized laboratories. Lately, immunnostaining methods have been described using PML antibodies for faster diagnosis of APL. The distinction between APL (AML-M3) and non AML-M3 AML is based upon microgranular versus speckled pattern observed in the nuclei of leukemic cells. Only few studies have described use of PML monoclonal antibody (Moab) – PG-M3. Typical flowcytometric (FCM) immunophenotype of APL cells reveals positivity for CD33 and CD13, with negativity for HLA-DR and CD34. RT-PCR (gold standard method) is used to detect classical APL genetic abnormality PML/RARa hybrid transcripts resulting from t(15;17)(q22;q21) and also the alternate translocations associated with APL. We undertook this study to assess the role of PML immunoflourescence (IF), flowcytometry and RT-PCR for quick diagnosis of APL. Patients and methodsDuring last 17 months, peripheral blood and/or bone marrow samples from 93 consecutive acute non-lymphocytic leukemia (ANLL) cases were obtained after informed consent. All the cases were classified using standard morphological criteria (FAB classification). RT-PCR for PML/RARa, FCM immunophenotyping (IP) (Moab panel comprising of CD13, CD33, CD34, HLA-DR, CD56, CD2, CD19, CD14 and CD64) and PML IF (using anti-PML Moab PG-M3 clone) were performed. In the PML IF study, staining pattern of leukemic cells was noted within 2 hours of staining, using Leica DM LB2 epifluorescence microscope equipped with chilled digital color camera and Leica FW 4000 software. In our experience PML-IF could be completed in 2-4 hrs, FCM-IP in 3-5 hrs and RT-PCR in 12-24 hrs. Results27/93 (29%) cases belonged to AML-M3 category and rest to different categories of AML (M0:1, M1:9, M2: 39, M4:10, M5:6, M6:1). RT–PCR for PML/RARa was positive in 24/27 (88.88%) cases with morphological diagnosis of AML-M3. bcr1 transcripts were detected in 2 (8.33%), bcr2 transcripts in 8 (33.33%) and bcr3 transcripts in 14 (58.33%) among 24 cases. 22/24 cases positive for PML/RARa by RT-PCR showed typical microgranular pattern of nuclear staining on PML IF (91.66% concordance). PML IF did not give any signal in one case and another one showed speckled pattern. In non AML-M3 cases, RT-PCR for PML-RARa was negative in all and PML immunoflourescence staining revealed speckled pattern in 59/66 (89.39%) cases. Typical FCM signature for APL was seen in 86.66% cases positive for PML/RARa by RT-PCR. Two cases were HLA DR positive and one out of these was positive for PML-RARa RT-PCR. ConclusionsImmunostaining with PML-antibody was found to be a rapid, simple, cost effective & less time consuming technique to detect PML-RARA with high rate of concordance with the gold standard RT-PCR. This could be routinely applied as an upfront investigation to pick up the APL cases. Disclosures:No relevant conflicts of interest to declare.

Continuous High-Dose Chlorambucil in Previously Untreated Patients with Chronic Lymphocytic Leukemia –Single-Center Experience.

Abstract 2372Poster Board II-349Chlorambucil is an effective and safe oral alkylating agent used for treatment of indolent lymphoproliferative disorders. While once considered a drug of choice for CLL, it has recently fallen out of favor, mostly due to clinical trials showing that fludarabine-based combinations have better disease-free, albeit not overall survival (OS). In these studies chlorambucil was administered once every 2-4 weeks. Results of some clinical trials suggest that continuous use of high doses of chlorambucil (≥ 10mg /day) is superior to intermittent administration. However, there are very few publications reporting outcomes of this type of treatment, especially outside of clinical trials. Here we summarize our experience with this approach in unselected patients with CLL. This was a retrospective study performed by chart-review of patients treated at our center. Patients were included in this analysis if they had CLL according to standard immunologic, morphologic and hematologic criteria, were in need of treatment and received chlorambucil at a daily dose of 10 mg or more for at least 4 weeks or until response or unacceptable toxicity as front-line treatment. Simultaneous administration of steroids but not other cytotoxic drugs was allowed. Use of low-dose chlorambucil maintenance (≤ 5 mg/day) after stopping high-dose chlorambucil was not considered treatment failure. The Kaplan-Meier method was used for creating survival curves and log-rank for comparisons of temporal variables. We identified 88 patients fulfilling the inclusion criteria, 70 men and 18 women, who were at the time of treatment start 38-88 years old (median 64). Nineteen patients were in Binet stage A, 38 B and 31 C; 2 were in Rai stage 0, 27 stage 1, 38 stage 2, 6 stage 3 and 25 stage 4. Twenty-nine had a low and 59 high total-tumor-mass index (TTM). The dose of chlorambucil ranged form 10 to 20 mg daily (median 15) and duration of treatment 2 to 20 wks (median 5). Forty-seven patients (53%) also received low-dose chlorambucil maintenance. The treatment was well tolerated. Three patients (3%) had serious infections, 2 died; 5 (6%) had severe hematological toxicity, mostly thrombocytopenia. Nausea, very common with pulse-doses of chlorambucil, was absent or mild; only 1 patient had severe nausea. Sixty-nine patients (78%) responded. After a median follow-up of survivors of 58 months, 26 patients are still alive and 17 have not failed treatment. Median OS was 53 months, 95% confidence-interval (CI) 40-66 months, time to treatment failure (TTF) 20 months (95% CI 13-27) (Fig. 1) and progression-free survival 15 months (95% CI 8-22). Age had no influence on treatment outcome. OS of patients with favorable stages (Rai 0-2, Binet A&B) was 60 months and of those with unfavorable stages 44 months. TTF was 30 and 11 months in these groups respectively. Both of these differences barely failed to reach statistical significance (p=0.067 and 0.058). TTM of 9 or higher was the only statistically significant negative prognostic factor that we were able to identify. OS was 78 vs. 44 months (p=0.021) and TTF 46 vs. 15 months (p=0.027). In conclusion, our results suggest that continuous administration of relatively high doses of chlorambucil is superior to intermittent administration, resulting in better efficacy and lower toxicity. While our results are inferior to those generally reported with fludarabine-based front-line treatments, continuous high-dose chlorambucil is less toxic, cheaper, the duration of treatment is shorter and OS is not different. This makes continuous high-dose chlorambucil an interesting approach for elderly, time- or financially constrained patients or health-care systems. Also, the low-toxicity and short treatment duration make it an interesting platform for the addition of non-toxic targeted drugs, like rituximab. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

In vitro maturation (IVM) of murine and human germinal vesicle (GV) stage oocytes using variations of a human fallopian tube epithelial feeder cell system

OBJECTIVE: To assess the maturation rate of murine and human GV oocytes using variations of a human tubal feeder cell system. DESIGN: Murine and human GV oocytes were randomized to human tubal fluid (HTF) media alone or co-culture with primary (CP58) or immortalized (CP13IM) human tubal cells. Rates of maturation were compared between groups. MATERIALS AND METHODS: Tubal epithelial cells were isolated from salpingectomy specimens by digestion with DNase/Collagenase. One batch was immortalized (CP13IM). Mouse GV oocytes were isolated from 8 week-old mice 40 h after PMSG stimulation. Human GV oocytes (n=21) were obtained from the MGH IVF lab. Murine and human GV oocytes were cultured in HTF media alone or on feeder layers of CP58 and CP13IM cells. Maturation rates were assessed by standard morphological criteria and compared between groups. RESULTS: Maturation rates of GV oocytes under the various culture conditions are shown in table 1. Tabled 1Maturation stage (% of total) of GV oocytes with different culture conditionsMurine (n=210, 24 h)Human (n=21)Culture systemGVGVBDMIIMII (24 h)MII (48 h)HTF27.5021.2551.2512.537.5CP588.7513.7577.5062.575.0CP13 IM12.5017.5070.0060.060.0 Open table in a new tab For murine GV oocytes (n=210 in 3 independent experiments), the maturation rate to the MII stage was approximately 50% higher with CP58 cells and nearly 40% higher with CP13IM cells compared to media alone. For human GV oocytes, the rate of maturation to the MII stage was approximately 5-fold higher with both the CP58 and CP13IM feeder systems at 24 h and approximately 2-fold higher at 48 h. CONCLUSIONS: This study shows that co-culture with either primary or immortalized tubal cells improved IVM rates for murine and human GV oocytes, suggesting a promising new technique with future applications in clinical reproductive medicine or in basic science research, such as in the generation of new stem cell lines.

Biochemical, Functional, and Pharmacological Characterization of AT-56, an Orally Active and Selective Inhibitor of Lipocalin-type Prostaglandin D Synthase

We report here that 4-dibenzo[a,d]cyclohepten-5-ylidene-1-[4-(2H-tetrazol-5-yl)-butyl]-piperidine (AT-56) is an orally active and selective inhibitor of lipocalin-type prostaglandin (PG) D synthase (L-PGDS). AT-56 inhibited human and mouse L-PGDSs in a concentration (3–250 μm)-dependent manner but did not affect the activities of hematopoietic PGD synthase (H-PGDS), cyclooxygenase-1 and -2, and microsomal PGE synthase-1. AT-56 inhibited the L-PGDS activity in a competitive manner against the substrate PGH2 (Km = 14 μm) with a Ki value of 75 μm but did not inhibit the binding of 13-cis-retinoic acid, a nonsubstrate lipophilic ligand, to L-PGDS. NMR titration analysis revealed that AT-56 occupied the catalytic pocket, but not the retinoid-binding pocket, of L-PGDS. AT-56 inhibited the production of PGD2 by L-PGDS-expressing human TE-671 cells after stimulation with Ca2+ ionophore (5 μm A23187) with an IC50 value of about 3 μm without affecting their production of PGE2 and PGF2α but had no effect on the PGD2 production by H-PGDS-expressing human megakaryocytes. Orally administered AT-56 (<30 mg/kg body weight) decreased the PGD2 production to 40% in the brain of H-PGDS-deficient mice after a stab wound injury in a dose-dependent manner without affecting the production of PGE2 and PGF2α and also suppressed the accumulation of eosinophils and monocytes in the bronco-alveolar lavage fluid from the antigen-induced lung inflammation model of human L-PGDS-transgenic mice.

The effect of media pH on embryo development and cycle outcome

OBJECTIVE: There has been debate on the optimal embryo culture conditions during in vitro fertilization cycles. This study was undertaken to determine the effect of media pH on embryo quality, the development of early cleaving embryos, and cycle outcome. DESIGN: Prospective, non-randomized study. MATERIALS AND METHODS: Fifteen patients with a minimum of 15 oocytes at retrieval were included in the study. Study patients had their oocytes split between a test incubator with an increased CO2 concentration and a control incubator set at a standard CO2 level. This study design allowed for the patient to act as their own control group. The oocytes from both incubators were observed for fertilization, the presence of early cleaving embryos, and Day 3 development at set hours. The embryos were graded using a standard morphology assessment protocol in order to choose the best quality embryos for Day 3 transfer regardless of which incubator was used for culture. Embryos that were of good quality and not chosen for transfer were subsequently frozen on Day 3. Patients were monitored post transfer and a positive pregnancy defined as the presence of an intrauterine sac. Data were analyzed using the unpaired t-test and the Fisher's exact test, with significance set at P < 0.05. RESULTS: The average pH values of the test and control incubators were significantly different (7.30 vs. 7.38, p < 0.0001). The lowered pH did result in a significantly higher number of blastomeres on Day 3 (7.11 vs. 6.25, p = 0.025). In addition, a higher percentage of embryos transferred were derived from the test incubator (22.0% vs. 10.7%, p = 0.045). While there was a trend towards an increased percentage of early cleaving embryos from the test incubator (22.0% vs. 17.2%) the difference was not significant (p = 0.08). There was no difference in the fertilization rate, embryo fragmentation rate, the development of embryos to at least the five cell stage on Day 3, or the percentage of frozen and discarded embryos. Nine cycles were examined where all of the transferred embryos came from exclusively one incubator. The clinical pregnancy rate was determined to be identical in these two groups (33.3%). CONCLUSIONS: This study demonstrated the role that media pH has on embryo quality as assessed by standard morphological criteria. Ongoing studies are being performed to further establish the effect of decreased media pH on clinical outcomes.

The effect of angiotensin type 1 receptor blockade on adhesion molecules in patients with IgA nephropathy.

Sir, Various studies have linked inflammation and endothelial dysfunction in patients with chronic renal disease [1]. Plasma levels of adhesion molecules [soluble intracellular adhesion molecule-1 (sICAM) and soluble vascular adhesion molecule-1 (sVCAM)] are markers of endothelial dysfunction and also risk factors for cardiovascular disease in patients with IgA nephropathy (IgAN) [2]. It is known that vascular lesion begins long before its clinical manifestation and its pathogenesis involves endothelial dysfunction and low-grade inflammation. Numerous studies provide evidence that ACE inhibitors or angiotensin II receptor antagonists (ARBs) are more effective than other antihypertensive drugs in slowing the progressive decline in glomerular filtration rate in IgAN [3]. Our aim was to test whether blocking the renin–angiotensin system (RAS) with irbesartan decreases levels of adhesion molecules in patients with biopsy-proven IgAN. We included in our study 36 patients (M/F 26/10, 51 ± 12.5 years). The inclusion criteria were biopsy-proven IgAN (defined by standard morphologic and immunohistochemical criteria), serum creatinine ≤1.5 mg/dl and urinary protein excretion ≥500 mg/day in at least three consecutive determinations during the previous 6-month period. Exclusion criteria were diabetes mellitus, coronary artery disease, peripheral vascular disease, stroke, acute infection or the inflammatory process on course and marked hypercholesterolaemia. Blood samples were collected from all patients before (T0) and after 16 weeks (T1) of treatment with 300 mg of irbesartan given once daily in the morning. A thorough blood chemistry control was performed in all patients. Creatinine clearance was determined by using venous blood for serum levels of creatinine and a 24-h urine collection. Serum and urinary creatinine concentration was measured using the Jaffe method. Serum intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 were measured by immunosorbent assay (ELISA). Statistical analyses were performed using SPSS version 13.0 (SPSS, Chicago, IL, USA). Systolic (SBP) and diastolic blood pressure (DBP) was significantly lower after irbesartan was given (from SBP T0: 144 ± 19 mmHg DBP T0: 93 ± 9 mmHg to SBP T1: 129 ± 9 mmHg DBP T1: 88 ± 8 mmHg, P < 0.01). Proteinuria levels were also significantly lower after treatment: from 1.6 ± 0.7 g/24 h − T0 to 1.1 ± 0.9 g/24 h − T1 (P < 0.001). We observed a significant decrease of sICAM and sVCAM plasma levels in patients after treatment with irbesartan (sICAM T0: 628 ± 163 ng/ml to sICAM T1: 369 ± 112 ng/ml, P < 0.001; sVCAM T0: 1028 ± 649 ng/ml to sVCAM T1: 688 ± 248 ng/ml, P < 0.001) (Table ​(Table11). Table 1 Summary of before (T0) and (T1) the treatment in each parameter (BP, sICAM and sVCAM) Studies [2,4] have proven the important role of inflammation in the outcome of IgAN. Angiotensin type 1 (AT1) receptor antagonist significantly decreases proteinuria and slows renal deterioration in patients with IgAN. Our data suggest that treatment with irbesartan in patients with IgAN has a beneficial effect on inflammatory markers. The interfering with the inflammatory markers of AT1 antagonist could, at least partially, explain the effect of AT1 receptor blockade on renal survival in patients with IgAN. It is obvious that additional studies are needed to verify this hypothesis, especially in order to rule out the direct effect of lowering blood pressure on the inflammatory markers taken into consideration. Conflict of interest statement. None declared.

Endoscopic Elastosonography Is Highly Predictive of Definite Pathology

Background: Elastosonography is a technique that allows for real-time assessment of elasticity or stiffness of tissue with the aid of conventional ultrasound instruments. In recent years this technique has been developed for endoscopic ultrasound (EUS). Preliminary data show that it might be a useful adjunct to standard morphological criteria to define a lesion without FNA sampling, or to select the optimal location for FNA to increase the yield of tissue sampling. To further elucidate this hypothesis we evaluated the performance of elastosonography in series of patients referred for EUS. Methods: patients were examined by an experienced endosonographer with a linear echoendoscope (Pentax 3830-UT) combined with a Hitachi EUB-8500 system to allow for realtime processing of elastonography images. Based on these images lesions were classified as either benign, malignant or indeterminate based on previously published data. A definite diagnosis was made on results of either EUS-FNA, surgical pathology or clinical follow-up (at least six months). Results: 41 procedures were performed in the same number of patients (M/F 17/24, age range 26 to 74 yrs): 25 procedures were done for mediastinal pathology, 13 for pancreatic masses, 1 each for rectal, adrenal and submucosal lesions. Elastosonograpy images were interpreted as benign in 10 cases (24%), indeterminate in 7 cases (17%), and malignant in 24 cases (59%). All patients with a benign result on elastosonography had benign disease during follow-up and EUS-FNA. Of the patients with lesions that were judged to be indeterminate on elastosonography (n = 7; 17%) 5 had benign disease based on clinical follow-up and EUS-FNA whereas 2 had malignant disease based on EUS-FNA. In the group with malignant elastosonography (n = 24) one patient had benign disease. A comparison of the elastography classes benign plus indeterminate versus malignant yielded a sensitivity of elastonosonography of 92%, a specificity of 94%, and negative and positive predictive values of respectively 96% and 88%. Conclusion: Elastosonography is highly sensitive and specific in determining the nature of both mediastinal and pancreatic lesions with excellent positive and negative predictive values and can therefore be helpful in guiding EUS-FNA and patient management.

Application of Immunohistochemistry to Thyroid Neoplasms

Thyroid lesions with nodular architecture and follicular pattern of growth often pose difficulties in accurate diagnosis during the assessment of cytologic and histologic specimens. The diagnosis of follicular neoplasm on cytology or of follicular tumor of uncertain malignant potential on histology is likely to cause confusion among clinicians and delay effective management of these lesions. Occasionally, thyroid tumors represent unusual or metastatic lesions and their accurate diagnosis requires immunohistochemical confirmation. To review the literature on the applications of immunohistochemistry in the differential diagnosis of thyroid tumors. Relevant articles indexed in PubMed (National Library of Medicine) between 1976 and 2006. Our review supports the use of ancillary techniques involving a panel of antibodies suitable for immunohistochemistry and molecular analysis in the assessment of thyroid nodules. These tools can improve diagnostic accuracy when combined with standard morphologic criteria.

Wertigkeit der konventionellen oralen Bürstenbiopsie

This study evaluated the performance of oral brush biopsies using standard morphological analysis and haematoxylin and eosin (HE) staining for detecting oral squamous cell carcinomas and their respective precursor lesions Brush biopsies were obtained in 169 consecutive patients who underwent routine biopsies and histological examination for clinically suspicious oral lesions. Air-dried smears were processed by acetone fixation and HE staining. Cytological assessment used well-established criteria of atypia to classify the specimen as either "tumor negative" (no signs of atypia, no malignant cells) or "tumor positive" (malignant cells, any sign of atypia or doubtful cells). Despite a sufficient number of cells, a definite cytological diagnosis could not be established in six cases. According to the criteria specified above, these specimens were classified as "tumor positive." The cytological analysis identified 49 out of 62 oral malignancies (sensitivity 79%). Seven out of 107 benign lesions were classified as false positive (specificity 93%). The positive and negative predictive values were each 88%. Oral brush biopsies will identify only about 80% of oral malignancies when the smears are processed by routine HE stains and are analysed via standard morphological criteria. Thus, this technique should not be used for diagnostic proof or to exclude malignant cells in a lesion suspicious for cancer. However, oral brush biopsy provides a versatile back-up strategy to uncover the true nature of the disease if a lesion is clinically considered benign by mistake.

Characteristic Expression Patterns of TCL1, CD38, and CD44 Identify Aggressive Lymphomas Harboring a MYC Translocation

The distinction between Burkitt (BL) or atypical Burkitt/Burkitt-like lymphomas harboring a MYC translocation (MYC+) and diffuse large B-cell lymphomas (DLBCLs) with high proliferation fractions but without a MYC translocation (MYC-) can be difficult using standard morphologic and immunohistochemical criteria. Recently, unique gene expression profiles differentiating BL and DLBCL were reported and include higher transcript levels of T-cell leukemia-1 (TCL1) and CD38 and lower transcript levels of CD44 in MYC+ BL relative to MYC- DLBCL. We examined a cohort of 67 cytogenetically defined aggressive lymphomas using immunohistochemical techniques for expression of TCL1, CD38, and CD44 and found distinct expression patterns between MYC+ and MYC- tumors. Furthermore, these markers are better predictors of MYC status than combined staining for CD10 and BCL2. Thus staining for TCL1, CD38, and CD44 are useful ancillary tests to identify B-cell tumors for which confirmatory cytogenetic and/or fluorescent in situ hybridization studies assessing the status of the MYC locus should be pursued.

Massive isolation of anamorphous ascomycete yeasts Candida oleophila from plant phyllosphere

Many years of research has confirmed a wide distribution of anamorphous ascomycete yeasts in the phyllosphere of diverse plants of the Moscow oblast. Based on the standard morphological and physiological criteria, on the results of restriction analysis of the 5.8S-ITS rDNA region, and on the sequencing of the D1D2 region of 26S rDNA, these yeasts were identified as Candida oleophila Montrocher. Previous isolation of this species has been rare, possibly due to its incorrect identification. This species, together with phytobiotic basidiomycete yeasts, was shown to be dominant in the yeast epiphytic communities on the surface parts of plants. The relative abundance of C. oleophila is highest on plant fruits and increases significantly by the end of the vegetation period. Wide occurrence of this yeast species on fruits and in the phyllosphere may be related to its ability to compete with rapidly growing phytopathogenic fungi.

Tuning of the Outer Hair Cell Motor by Membrane Cholesterol

Cholesterol affects diverse biological processes, in many cases by modulating the function of integral membrane proteins. We observed that alterations of cochlear cholesterol modulate hearing in mice. Mammalian hearing is powered by outer hair cell (OHC) electromotility, a membrane-based motor mechanism that resides in the OHC lateral wall. We show that membrane cholesterol decreases during maturation of OHCs. To study the effects of cholesterol on hearing at the molecular level, we altered cholesterol levels in the OHC wall, which contains the membrane protein prestin. We show a dynamic and reversible relationship between membrane cholesterol levels and voltage dependence of prestin-associated charge movement in both OHCs and prestin-transfected HEK 293 cells. Cholesterol levels also modulate the distribution of prestin within plasma membrane microdomains and affect prestin self-association in HEK 293 cells. These findings indicate that alterations in membrane cholesterol affect prestin function and functionally tune the outer hair cell.

Cystic Fibrosis Transmembrane Conductance Regulator-independent Phagosomal Acidification in Macrophages

It was reported recently that the cystic fibrosis transmembrane conductance regulator (CFTR) is required for acidification of phagosomes in alveolar macrophages (Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., Huang, P., Tong, J., Naren, A. P., Bindokas, V., Palfrey, H. C., and Nelson, D. J. (2006) Nat. Cell Biol. 8, 933-944). Here we determined whether the CFTR chloride channel is a generalized pathway for chloride entry into phagosomes in macrophages and whether mutations in CFTR could contribute to alveolar macrophage dysfunction. The pH of mature phagolysosomes in macrophages was measured by fluorescence ratio imaging using a zymosan conjugate containing Oregon Green(R) 488 and tetramethylrhodamine. Acidification of phagolysosomes in J774A.1 macrophages (pH approximately 5.1 at 45 min), murine alveolar macrophages (pH approximately 5.3), and human alveolar macrophages (pH approximately 5.3) was insensitive to CFTR inhibition by the thiazolidinone CFTR(inh)-172. Acidification of phagolysosomes in alveolar macrophages isolated from mice homozygous for DeltaF508-CFTR, the most common mutation in cystic fibrosis, was not different compared with that in alveolar macrophages isolated from wild-type mice. We also measured the kinetics of phagosomal acidification in J774A.1 and murine alveolar macrophages using a zymosan conjugate containing fluorescein and tetramethylrhodamine. Phagosomal acidification began within 3 min of zymosan binding and was complete within approximately 15 min of internalization. The rate of phagosomal acidification in J774A.1 cells was not slowed by CFTR(inh)-172 and was not different in alveolar macrophages from wild-type versus DeltaF508-CFTR mice. Our data indicate that phagolysosomal acidification in macrophages is not dependent on CFTR channel activity and do not support a proposed mechanism for cystic fibrosis lung disease involving defective phagosomal acidification and bacterial killing in alveolar macrophages.

Limited importance of pre-embryo pronuclear morphology (zygote score) in assisted reproduction outcome in the absence of embryo cryopreservation

To investigate the hypothesis that Z-score criteria represent a reliable predictor of implantation rate and pregnancy outcome in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cycles, excluding the possibility of embryo selection before the embryo transfer. Retrospective clinical study. Centre of Reproductive Medicine, Department of Obstetrics and Gynecology, Arcispedale S. Maria Nuova (ASMN), Reggio Emilia, Italy. We analyzed 393 pregnancies obtained by IVF or ICSI cycles. Morphologic evaluations of Z-score in pre-embryos obtained from IVF or ICSI cycles. Evaluations of Z-scores, implantation rate, and clinical pregnancy outcome. We did not find any statistically significant correlation between the Z-score of 1032 embryos transferred in 393 embryo transfers and the implantation rate or the pregnancy outcome. In particular, the best Z-score identified (Z1, 7.2%) did not seem to correlate with embryo implantation rate or pregnancy outcomes any better than those with worse scores (Z2, 6.9% and Z3, 85.9%). Our results seem to confirm that Z-score alone cannot be considered a better tool than standard morphologic criteria for identifying, controlling, or selecting embryos with a better chance of successful ongoing pregnancy.

Differentiation between Diverticulitis and Colorectal Cancer: Quantitative CT Perfusion Measurements versus Morphologic Criteria—Initial Experience

To determine whether computed tomographic (CT) perfusion measurements in prospectively recruited patients can be used to differentiate between diverticulitis and colorectal cancer and to compare this discrimination with that of standard morphologic criteria. After institutional review board approval and written informed consent were obtained, 60 patients (24 men, 36 women; mean age, 69 years; range, 33.5-90.4 years; 20 patients with cancer, 20 with diverticulitis, and 20 with inactive diverticular disease) underwent CT perfusion imaging at the level of the colonic abnormality, and perfusion parameters were calculated. Analysis of variance was used to investigate any differences in perfusion between the patient groups. Two independent observers also analyzed an abdominopelvic CT study obtained immediately after the CT perfusion study and noted standard morphologic criteria for differential diagnosis. The sensitivity and specificity of CT perfusion measurements for determining the diagnostic category were compared with morphologic criteria by means of multivariate analysis to identify the most discriminatory criteria. Mean blood volume, blood flow, transit time, and permeability were significantly different between patients with cancer and those with diverticulitis (P < .0001); patients with cancer had the highest blood volume, blood flow, and permeability and the shortest transit time. The most discriminatory criteria for determining diagnostic category were blood volume, transit time, permeability, and presence of pericolonic nodes (P = .05, .02, .04, and .02, respectively). Blood volume and blood flow each had a sensitivity of 80% and had specificity of 70% and 75%, respectively, for cancer in comparison with standard morphologic criteria: less than 5 cm of bowel involvement (45% sensitivity, 95% specificity), presence of a mass (85% sensitivity, 90% specificity), pericolonic inflammation (75% sensitivity, 5% specificity), and pericolonic nodes (90% sensitivity, 45% specificity). CT perfusion measurements enable differentiation and better discrimination, in comparison with morphologic criteria, between cancer and diverticulitis.