Pitfalls and Caveats in Applying Chromogenic Immunostaining to Histopathological Diagnosis

Chromogenic immunohistochemistry (immunostaining using an enzyme-labeled probe) is an essential histochemical technique for analyzing pathogenesis and making a histopathological diagnosis in routine pathology services. In neoplastic lesions, immunohistochemistry allows the study of specific clinical and biological features such as histogenesis, behavioral characteristics, therapeutic targets, and prognostic biomarkers. The needs for appropriate and reproducible methods of immunostaining are prompted by technical development and refinement, commercial availability of a variety of antibodies, advanced applicability of immunohistochemical markers, accelerated analysis of clinicopathological correlations, progress in molecular targeted therapy, and the expectation of advanced histopathological diagnosis. However, immunostaining does have various pitfalls and caveats. Pathologists should learn from previous mistakes and failures and from results indicating false positivity and false negativity. The present review article describes various devices, technical hints, and trouble-shooting guides to keep in mind when performing immunostaining.

Histochemical Distribution of Acetylcholinesterase in the Forebrain Nuclei of an Indian Non Catfish Channa punctatus

Background: Acetylcholinesterase (AChE) is an enzyme belonging to hydrolase group which splits the acetylcholine in to choline and acetate. It is supposed to be a marker of cholinergic and cholinoceptive neurons. Acetylcholinesterase histochemisry has been done in a number of vertebrates but it is still obscure and scattered in fishes, particularly in Indian fishes. Methods: In the present study a modified histochemical technique has been employed to histochemically map the acetylcholinesterase containing neurons in the telencephalic and diencephalic nuclei of C. punctatus described by Hedreen, et al (1985).Result: Acetylchoinesterase is differentially expressed in the various prosencephalic centres and nuclei of the brain, thus its staining clearly demarcates these centres and nuclei based on varying enzyme intensity. Among the pallial nuclei of the forebrain, medial and dorsolateral nuclei showed intense enzyme activity while ventral dorsolateral nucleus and central nucleus showed moderate reaction. In contrast, most of the subpallial nuclei of the forebrain showed high intensity. Diencephalic nuclei of the forebrain exhibited mosaic pattern of enzyme distribution.

Pitfalls and Caveats in Applying Immunostaining to Histopathological Diagnosis

Immunostaining is an essential histochemical technique for analyzing pathogenesis and making a histopathological diagnosis. The needs are prompted by technical development and refinement, commercial availability of a variety of antibodies, deepened knowledge of immunohistochemical markers, accelerated analysis of morphofunctional correlations, progress in molecular target therapy, and the expectation of advanced histopathological diagnosis. However, immunostaining does have various pitfalls and caveats. We should learn from mistakes and failures, as well as from false positivity and false negativity. The present review article describes various devices, technical hints and trouble-shooting guides to keep in mind in performing immunostaining.

Is Senescence-Associated β-Galactosidase a Reliable in vivo Marker of Cellular Senescence During Embryonic Development?

During vertebrate embryonic development, cellular senescence occurs at multiple locations. To date, it has been accepted that when there has been induction of senescence in an embryonic tissue, β-galactosidase activity is detectable at a pH as high as 6.0, and this has been extensively used as a marker of cellular senescence in vivo in both whole-mount and cryosections. Such senescence-associated β-galactosidase (SA-β-GAL) labeling appears enhanced in degenerating regions of the vertebrate embryo that are also affected by programmed cell death. In this sense, there is a strong SA-β-GAL signal which overlaps with the pattern of cell death in the interdigital tissue of the developing limbs, and indeed, many of the labeled cells detected go on to subsequently undergo apoptosis. However, it has been reported that β-GAL activity at pH 6.0 is also enhanced in healthy neurons, and some retinal neurons are strongly labeled with this histochemical technique when they begin to differentiate during early embryonic development. These labeled early post-mitotic neurons also express other senescence markers such as p21. Therefore, the reliability of this histochemical technique in studying senescence in cells such as neurons that undergo prolonged and irreversible cell-cycle arrest is questionable because it is also expressed in healthy post-mitotic cells. The identification of new biomarkers of cellular senescence would, in combination with established markers, increase the specificity and efficiency of detecting cellular senescence in embryonic and healthy mature tissues.

MORPHOLOGICAL RESEARCH ON FREE-RESIDUE OXIDATION PROCESSES IN CASES OF DECIDUAL CELLS OF PLACENTA IN CHORIOAMNIONOTIS AND BASAL DECIDUITIS COMBINED WITH IRON-DEFICIENCY ANEMIA IN THE PREGNANT

Background. The oxidative modification of proteins is lately pivotal to pathologists and it is a new way of research on different pathological conditions, as well as the diagnostics of inflammation processes in placenta.Objective. The study was aimed at the research of nitro peroxides and establishing the specific features of oxidative modification of proteins in inflammation of placenta with iron deficient anaemia in the pregnant.Methods. Сhemiluminescent and histochemical technique (with bromphenol blue on ‘acidic’ and ‘basic’ proteins according to Mikel Calvo) was applied.Results. The intensity of nitro peroxides glow in chorioamnionitis and basal deciduitis increased in comparison with the samples of physiological and iron deficient anaemia gestation. At the same time in chorioamnionitis the glow intensity is higher than in basal deciduitis.Due to the results of immune histochemical technique held while analysing the samples, together with chorioamnionitis and basal deciduitis the R/B increases and in basal deciduitis the rate, is probably, higher, than in chorioamnionitis. At the same time, the extent of oxidative modification of proteins in cases of inflammation with iron deficient anaemia in the pregnant is on the average higher than with no iron deficient anaemia in the pregnant.Conclusions. High level of nitro peroxides in placentae basal plate in secundines inflammation, the increase in R/B rate, in other words the prevalence of ‘acidic’ proteins over ‘basic’ ones, is evidenced due to the increase of the intensity of oxidative modification processes of proteins in cases of deciduitis.

Myology

Diseases of muscle have become better understood by careful clinical observations, resulting in a clinically useful classification of the different groups of disorders e.g. inherited muscular dystrophies such as Duchenne muscular dystrophy, limb-girdle and metabolic myopathies, and myotonic disorders. A number of scientific approaches have determined the directions taken by this evolving classification. Understanding of the anatomy of the motor unit’s distribution in muscle transformed muscle pathology and muscle electrophysiology, and key to these pathological advances was the use of the histochemical technique for identifying myofibrillar ATPase in muscle fibres. This allowed studies of the distribution of fibre types in muscle in many different disorders. The inflammatory muscle diseases have been better understood since recent advances in immunology have characterized the underlying processes. The limb-girdle and childhood myopathies have proven to be heterogeneous, with many different, apparently causative, underlying genetic mutations.

Evaluation of EnSeal®, an adaptive bipolar electrosurgical tissue-sealing device

Relatively few, and inconsistent, data are available in the literature about the properties of EnSeal®, an electrosurgical tissue-sealing device. For this reason, we conducted control safety tests on experimental pigs. The mean burst pressure of sealed vessels (2–7 mm in diameter) proved to be 873.89 ± 120.57 mmHg (n = 60). Surface temperature increased to 69.25 ± 0.98 °C in average (n = 22). The mean diameter of the collateral microscopic thermal injury zone was found to be 0.28 ± 0.04 mm, and it did not show significant differences among the groups of tissues studied (n = 183). During our studies, the device worked reliably and met the relevant requirements in all cases. It can be established that EnSeal® enables high-safety clinical interventions at high blood pressure values, in different tissues and even at sites adjacent to heat-sensitive tissues, and thus it paves the way for new operative solutions in both human and veterinary surgery. In our opinion, the discrepancies between data reported in the literature arise from differences in the design of studies and in the designated limit values. To ensure standardisation, we recommend the use of the nitroblue-tetrazolium chloride/lactate dehydrogenase (NBTC/LDH) enzyme histochemical technique for studying thermal injury induced by the different performance levels and application times of devices operating with electromagnetic energy.