Use of fine needle aspirate from peripheral nerves of pure-neural leprosy for cytology and PCR to confirm the diagnosis: A pilot study.

Background: The diagnosis of pure neural leprosy (PNL) remained subjective because of over-dependence of clinical expertise and a lack of simple yet reliable diagnostic tool. The criteria for diagnosis, proposed by Jardim et al., are not routinely done by clinicians in developing country as it involves invasive nerve biopsy and sophisticated anti-PGL-1 detection. We conducted a study using fi ne needle aspiration cytology (FNAC) coupled with Ziehl Neelsen staining (ZN staining) and Multiplex- Polymerase Chain Reaction (PCR) specifi c for M. leprae for an objective diagnosis of pure neural leprosy (PNL), which may be simpler and yet reliable. Aim: The aim of the study is to couple FNAC with ZN staining and multiplex PCR to diagnose pure neural leprosy patients rapidly, in simpler and yet reliable way. Methods: Thirteen patients of PNL as diagnosed by two independent consultants were included as case, and 5 patients other than PNL were taken as control in the study. Fine needle aspiration was done on the affected nerve, and aspirates were evaluated for cytology, ZN staining and multiplex- PCR. Results: Out of the 13 cases where fi ne needle aspiration was done, M. leprae could be elicited in the nerve tissue aspirates in 5 cases (38.4%) with the help of conventional acid-fast staining and 11 cases (84.6%) with the help of multiplex PCR. On cytological examination of the aspirates, only 3 (23%) cases showed specifi c epithelioid cells, whereas 8 (61.5%) cases showed non-specifi c infl ammation, and 2 (15.3%) cases had no infl ammatory cells. Conclusion: Our study demonstrates that in the fi eld of laboratory diagnosis of PNL cases, FNAC in combination with ZN staining for acid-fast bacilli (AFB) and Multiplex-PCR can provide a rapid and defi nitive diagnosis for the majority of PNL cases. FNAC is a less-invasive, outdoor-based and simpler technique than invasive nerve biopsy procedure. Thus, this study may enlighten the future path for easy and reliable diagnosis of PNL.

Green fl uorescent protein: a molecular lantern that illuminates the cellular interior.

The green fl uorescent protein (GFP) has become an extremely popular reporter molecule in contemporary cell biology research in a relatively short span of time (fi gure 1). GFP fl uorescence is extensively used as a tool for monitoring gene expression, localisation, mobility, traffi c, and interaction of various membrane and cytoplasmic proteins. The major advantage of GFP as a fl uorophore is its intrinsic, cofactor-independent fl uorescence, which exhibits remarkable stability in the presence of denaturants and over a wide range of pH (Tsien 1998). In addition to visualisation by fl uorescence microscopy, GFP-tagged proteins can be monitored by a variety of techniques such as fl uorescence resonance energy transfer (FRET), fl uorescence recovery after photobleaching (FRAP) and fl uorescence correlation spectroscopy (FCS). The Nobel Prize in chemistry in 2008 was awarded for the discovery and development of GFP to Osamu Shimomura, Martin Chalfi e and Roger Tsien. GFP was discovered by Shimomura in the jellyfi sh Aequorea victoria (fi gure 2) in the early 1960s (Shimomura et al. 1962). It took almost three decades for the gene that encodes GFP to be cloned (Prasher et al. 1992). Subsequently, it was shown by Martin Chalfi e and co-workers that GFP could be expressed in heterologous systems (Chalfi e et al. 1994). This Figure 1. Number of publications (year-wise) in which the words green fl uorescent protein (GFP) appear either in the title or abstract (data taken from PubMed). This list is not exhaustive.

Chondroid lipoma--a case report.

Chondroid lipoma is a rare fatty tumor of soft tissues, especially in limbs and limb girdles. Though it is clinically benign, the main importance lies in its histological similarity with myxoid liposarcoma and chondrosarcoma, which have poorer prognosis. In our study, classical histological pattern of chondroid lipoma was confirmed on H&E and PAS stains with low mitotic count.

Skin cancers in chronic arsenic toxicity--a study of predictive value of some proliferative markers.

Prolonged exposure to arsenic contaminated water produces various clinical features, cutaneous features e.g. melanosis, keratosis and cancers being very common. Evaluation of such lesions by proliferative markers can provide useful information in regards to the biological behaviour of the lesions. Thus, cases with high proliferative status can be ominous sign for development of cancers. We studied skin biopsy of 42 cases. These were evaluated with AgNOR score and PCNA stain, in addition to H & E examination. Here, invasive cancer cases had mean AgNOR score of 3.56, those with severe dysplasia had 3.0, moderate and mild dysplasia scored 1.73, benign changes had mean score of 1.35 while normal control cases had 1.08. PCNA index in cancers was above 50, that of severe dysplasia 25-30, mild to moderate dysplasia 1.0-5.0, those with benign changes 0.5 -1.0 and normal control had LI of less than 0.5%. PCNA has the advantage of less chance of observer error over AgNOR stain.

Depth profiling in membranes by fluorescence quenching.

Membrane penetration depth is an important parameter in relation to membrane structure and organization. A methodology has been developed to analyze the membrane penetration depths of fluorescent molecules or groups utilizing differential fluorescence quenching caused by membrane embedded spin-label probes located at different depths. The method involves determination of the parallax in the apparent location of fluorophores, detected when quenching by phospholipids spin-labelled at two different depths is compared. By use of relatively simple algebraic expressions, the method allows calculation of depth in Å. This method has been used to determine the location of fluorophores in NBD-labelled lipids and anthroyloxy-labelled fatty acids in model membranes and of the membrane embedded tryptophan residues in the reconstituted nicotinic acetylcholine receptor.