Anthony Henwood

Current applications of orcein in histochemistry. A brief review with some new observations concerning influence of dye batch variation and aging of dye solutions on staining.

Current uses of orcein to demonstrate elastic fibers and, following permanganate oxidation (Shikatas modification), hepatitis B surface antigen, copper associated protein, and sulfated mucins, are reviewed. Variations in staining performance with batch of dye and age of dye solution is also discussed. Additional experimental findings support the view that the orcein stain for elastic tissue and Shikatas modification produces consistent, high quality results as long as appropriate controls and suitable dye batches, e.g., Biological Stain Commission certified dyes, are used.

Tape transfer sectioning of tissue microarrays introduces nonspecific immunohistochemical staining artifacts.

Abstract Tissue microarrays place tens to hundreds of formalin fixed, paraffin embedded tissue cores into a paraffin block in a systematic grid pattern that permits their simultaneous evaluation in a single section. The fragmented nature of the tissue cores often makes sectioning of tissue microarrays difficult so that the resulting disks of tissue lose their shape, fracture or fall out of the paraffin section altogether. We have evaluated an alternative sectioning protocol for stabilizing the tissue microarray surface by placing an adhesive tape “window” over the face of the paraffin block prior to sectioning. Once sectioned, the tape/sections are transferred directly onto coated microscope slides, thereby avoiding routine floating of sections on a water bath. After sectioning with either the tape transfer or standard protocols, slides were stained either using hematoxylin and eosin or immunohistochemistry using antibodies to S-100 protein and the tissue specific antigens, keratin (AE1/3) and the leukocyte common antigen CD45. We found that the tape method produced thicker sections that were darker and more densely packed with loss of tissue definition compared to sections prepared using water bath flotation. Quantitative image analysis of immunohistochemical staining demonstrated that the tape method produced a higher incidence of nonspecific staining, which raised the potential for false positive staining.

The application of heated detergent dewaxing and rehydration to immunohistochemistry

Abstract Hot commercial dishwashing detergent has been used to deparaffinize and hydrate formalin fixed, paraffin embedded sections for immunohistochemistry. Fifty-five antibodies, used routinely for diagnosis, were used to compare hot detergent dewaxing with the proprietary hydrocarbon-based dewaxing reagent supplied with the Bond Max immunohistochemistry system®. A 2% concentration of commercial dishwashing detergent in distilled water was heated to 90° C and paraffin sections were treated twice for 1 min each. Nearly all antibodies gave equivalent results except CD10 and CD57 (hydrocarbon-based dewaxing better) and CD45 and alpha fetoprotein (detergent dewaxing better); the differences, however, were minimal. There also was a significant cost saving using detergent dewaxing.

The application of heated detergent dewaxing and rehydration to techniques for the demonstration of fungi: a comparison to routine xylene-alcohol dewaxing

Abstract Fungal stains on slides dewaxed by domestic dishwashing detergent in hot water were compared to xylene and alcohol dewaxing. The fungal stains were Grocott’s Methenamine Silver (GMS), Periodic Acid Schiff’s following diastase (DPAS), and Sulphation Toluidine Blue (STB). For detergent dewaxing, a 2% concentration of domestic dishwashing detergent in distilled water heated to 90°C was used and paraffin sections were treated twice for 1 minute each. Hot detergent dewaxing is not only faster, cheaper, and safer than xylene dewaxing, but also equivalent or better than xylene for detecting fungi on DPAS, GMS and STB.

Fungal contamination of Hanks solution

Laboratory contamination of clinical specimens submitted for cytological examination is an on‐going threat that requires continuous vigilance and quality control. We recently were confronted with a suspected fungal infection in a bone aspirate that was found to derive from contaminated Hanks solution used in cytological preparation. Diff‐Quik staining of air‐dried cytocentrifuged preparations of Hanks fluid as well as any other suspect laboratory fluid is recommended as a routine quality control procedure. Diagn. Cytopathol. 2013.

Hematoxylin and eosin staining of mucins of the gastrointestinal tract

Abstract An infrequent observation of assessing hematoxylin and eosin sections is the blue staining of mucins (for example those in goblet cells). This is believed to be due to a low concentration of alum and high pH of the hematoxylin staining solution. This study examines the incidence of blue mucin in various sites of the gastrointestinal tract using a low alum, high pH hematoxylin solution. The results are compared with a conventional hematoxylin solution, iron alum celestine blue method and an alcian blue (pH 2.5)-periodic acid-Schiff (AB-PAS) stain to characterize the type of mucin demonstrated. This study is the first to offer evidence that blue-stained mucin with low alum, high pH hematoxylin corresponds with carboxylated mucins as shown by the AB-PAS stain in the gastrointestinal tract. Iron alum celestine blue was also found to stain the mucin of a proportion of rectal biopsies and appendix as well as the carboxylated mucin of one duodenal biopsy.

Diagnosis of pediatric genitourinary embryonal rhabdomyosarcoma by urine cytology: a case report.

BACKGROUND Urinary cytology is an uncommon investigative tool for pediatric tumors. CASE A 16-month-old infant presented for investigation ofhematuria and blood clots into diapers. Urinary cytology showed a population of small, round, blue cells with little cytoplasm and high nuclear/cytoplasmic ratios. The cells were positive for desmin and negative for cytokeratin. A cytologic diagnosis of embryonal rhabdomyosarcoma was offered and subsequently confirmed by histopathology. CONCLUSION Embryonal rhabdomyosarcomas are not uncommon in the pediatric population. Urinary cytology may be useful for rapid diagnosis and early management of such cases.

Improving placental block morphology using microwave-assisted fixation

ABSTRACT The histopathologic assessment of placenta is often marred by poorly fixed and processed sections. An audit conducted in the department resulted in 21% of placental blocks showing features of poor processing. A microwave-assisted fixation procedure was introduced using the Micromed KOS microwave oven (ABACUS-ALS, Brisbane, Australia) for 2 h at 45°C. This resulted in an improvement in the quality of the sections with a zero failure rate on a subsequent audit.

Looks like fungi but it isn’t – identifying pseudo-fungi*

Abstract Pseudo-fungi are structures that look like true fungi. They often give a positive periodic acid-Schiff and a negative Grocott’s methenamine silver reaction. From a therapeutic point of view, it is important to correctly characterize these structures as one or the other. This review presents guidelines for their accurate histochemical diagnosis.

Abstract 42: Expression of interleukin-7 and its signalling intermediates in human neuroblastoma tumours.

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Neuroblastoma (NB) is the most common extracranial childhood malignant solid tumour which shows remarkable biological heterogeneity. It has been speculated that schwann cells influence NB tumour growth, by secreting as yet to be identified ‘soluble factors’ that serve as anti-proliferative and differentiation signals for neuronal cells. Several gene expression studies indicate that interleukin 7 (IL7) has increased expression in NB specifically in tumours with a better prognosis, making IL7 a key candidate for this soluble factor. In this study, the expression of IL7, IL7 receptor (IL7R) and its downstream signalling proteins, including the Janus Kinases (JAK1 and JAK3), Signal Transducers and Activators of Transcription (STAT5) and Phosphorylated STAT5 (pSTAT5) were analysed using immunohistochemical stains in a cohort of 100 patients with stroma poor NB and 24 with stroma rich ganglioneuroma (GN). The expression of these proteins were correlated to the expression of the diagnostic markers S100 (stromal marker), NB84 (neuroblast marker) and CD99 (negative marker for NB). The immunohistochemical analysis showed that IL7 expression was strongly positive exclusively in schwannian stroma in both favourable and unfavourable histology whilst staining ganglionic cells as well. The levels of expression, as quantified by specialised digital image analysis algorithms (Aperio, USA), revealed IL7, IL7R, JAK1, STAT5 and pSTAT5 were all increased in GNs compared to NBs. There was no correlated change in expression levels of JAK3 in the tumours. Expression of pStat5 was found to be significantly reduced (t-test, p=0.00013) in stroma poor NB compared to GN. Our findings implicate IL7 within the schwannian stroma of the tumour architecture as having a paracrine signalling effect on neighbouring neuroblasts which may provide the anti-proliferative and differentiation signals postulated. Further investigation of the importance of IL7 in neuroblastoma will define the biological significance of the IL7-Stat5 signalling cascade and whether targeting the specific IL7 signalling pathway proteins will raise novel treatment strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 42. doi:1538-7445.AM2012-42