Simultaneous cell morphometry and refractive index measurement with dual-wavelength digital holographic microscopy and dye-enhanced dispersion of perfusion medium.

Digital holographic microscopy (DHM) allows optical-path-difference (OPD) measurements with nanometric accuracy. OPD induced by transparent cells depends on both the refractive index (RI) of cells and their morphology. This Letter presents a dual-wavelength DHM that allows us to separately measure both the RI and the cellular thickness by exploiting an enhanced dispersion of the perfusion medium achieved by the utilization of an extracellular dye. The two wavelengths are chosen in the vicinity of the absorption peak of the dye, where the absorption is accompanied by a significant variation of the RI as a function of the wavelength.

The impact of cyclin D1 mRNA isoforms, morphology and p53 in mantle cell lymphoma: p53 alterations and blastoid morphology are strong predictors of a high proliferation index.

BACKGROUND: Mantle cell lymphoma is a clinically heterogeneous disease characterized by overexpression of cyclin D1 protein. Blastoid morphology, high proliferation, and secondary genetic aberrations are markers of aggressive behavior. Expression profiling of mantle cell lymphoma revealed that predominance of the 3'UTR-deficient, short cyclin D1 mRNA isoform was associated with high cyclin D1 levels, a high "proliferation signature" and poor prognosis. DESIGN AND METHODS: Sixty-two cases of mantle cell lymphoma were analyzed for cyclin D1 mRNA isoforms and total cyclin D1 levels by real-time reverse transcriptase polymerase chain reaction, and TP53 alterations were assessed by immunohistochemistry and molecular analysis. Results were correlated with proliferation index and clinical outcome. RESULTS: Predominance of the short cyclin D1 mRNA was found in 14 (23%) samples, including four with complete loss of the standard transcript. TP53 alterations were found in 15 (24%) cases. Predominance of 3'UTR-deficient mRNA was significantly associated with high cyclin D1 mRNA levels (P=0.009) and more commonly found in blastoid mantle cell lymphoma (5/11, P=0.060) and cases with a proliferation index of >20% (P=0.026). Both blastoid morphology (11/11, P<0.001) and TP53 alterations (15/15, P<0.001) were significantly correlated with a high proliferation index. A proliferation index of 10% was determined to be a significant threshold for survival in multivariate analysis (P=0.01). CONCLUSIONS: TP53 alterations are strongly associated with a high proliferation index and aggressive behavior in mantle cell lymphoma. Predominance of the 3'UTR-deficient transcript correlates with higher cyclin D1 levels and may be a secondary contributing factor to high proliferation, but failed to reach prognostic significance in this study.

Comparative study of human erythrocytes by digital holographic microscopy, confocal microscopy, and impedance volume analyzer.

Red blood cell (RBC) parameters such as morphology, volume, refractive index, and hemoglobin content are of great importance for diagnostic purposes. Existing approaches require complicated calibration procedures and robust cell perturbation. As a result, reference values for normal RBC differ depending on the method used. We present a way for measuring parameters of intact individual RBCs by using digital holographic microscopy (DHM), a new interferometric and label-free technique with nanometric axial sensitivity. The results are compared with values achieved by conventional techniques for RBC of the same donor and previously published figures. A DHM equipped with a laser diode (lambda = 663 nm) was used to record holograms in an off-axis geometry. Measurements of both RBC refractive indices and volumes were achieved via monitoring the quantitative phase map of RBC by means of a sequential perfusion of two isotonic solutions with different refractive indices obtained by the use of Nycodenz (decoupling procedure). Volume of RBCs labeled by membrane dye Dil was analyzed by confocal microscopy. The mean cell volume (MCV), red blood cell distribution width (RDW), and mean cell hemoglobin concentration (MCHC) were also measured with an impedance volume analyzer. DHM yielded RBC refractive index n = 1.418 +/- 0.012, volume 83 +/- 14 fl, MCH = 29.9 pg, and MCHC 362 +/- 40 g/l. Erythrocyte MCV, MCH, and MCHC achieved by an impedance volume analyzer were 82 fl, 28.6 pg, and 349 g/l, respectively. Confocal microscopy yielded 91 +/- 17 fl for RBC volume. In conclusion, DHM in combination with a decoupling procedure allows measuring noninvasively volume, refractive index, and hemoglobin content of single-living RBCs with a high accuracy.

Sentinel nodes and and non sentinel nodes evaluation in T1 melanoma patients

Objective: Because increasing incidence of melanoma and dermatologicsystematic screening, more early superficial melanoma are discovered in Switzerland. Patients with Breslow index more than 1 mm. (T2) represent the classical indication to sentinel node (SN). It has been shown that some ''risky'' T1 patients may have micrometastatic SNs. T1b melanoma are defined by presence of ulceration,Clark IV (ormore) level, signs of melanoma regression (old classification) and high mitotic index (new TNM). The objective of the present study was to review the incidence and risk for metastatic SN in T1 patients and if radical lymph node dissection is justified (evaluation of non sentinel node [NSN]) compared with T2-4 patients.Methods: Retrospective review of a cohort of all patients operated for T1-4 clinically N0 and radiological M0 melanoma patients between 1997 and 2010 in a reference melanoma centre.Results: 599 melanoma patients have been operated with SNdissection. There were 98 T1 patients. Metastatic SN were observed in 2 out of 24 T1a patients and in 5 out of 74 T1b patients. This means overall 7% T1 patients were at least N1a. None of SN+ T1a or T1b patients had metastatic NSN after radical lymph node dissections (RLND). During the follow-up (1998-2010), no patients presented with locoregional disease and only one T1a N1a patient died of metastatic melanoma. These results contrast with the other 591 T2-4 patients: 150 were SN+ (25%) and among them 23 had metastatic NSN after RLND. Overall 23/136 (17%) had metastatic NSN.Conclusion: T1 melanoma patients are at significant risk (7%) for metastatic lymph node in the corresponding drainage basin. T1a and T1b did not differ regarding this risk. However, the benefit for a RLND must be reevaluated regarding surgical morbidity, because none of T1 patients had metastatic NSN.