Acetylcholinesterase histochemistry of rectal suction biopsies in the diagnosis of Hirschsprung's disease.

Rectal suction biopsy with acetylcholinesterase (AChE) histochemistry has gained increased acceptance as the means of definitely diagnosing Hirschsprung's disease (HD) as well as of excluding this diagnosis when evaluating children with low intestinal obstruction or chronic constipation since the report of Meier-Ruge et al. in 1972. But this AChE histochemical study has not been reported yet in Korea. During the 14-month period from April, 1991 through June, 1992, 37 children, aged 3 days to 17 years had rectal suction biopsies for the diagnosis or exclusion of HD. In this study, AChE histochemistry (N = 37) was compared with hematoxylin & eosin (H&E) staining of same suction biopsy specimens (N = 35) for diagnostic accuracy. The histochemical criterion used for the diagnosis of Hirschsprung's disease was that of Chow et al. (1977), i.e., the presence of many coarse discrete cholinergic fibers in the muscularis mucosae and in the immediately subjacent submucosa regardless of an infiltration of cholinergic fibers in the lamina propria. Of 13 biopsies from the patients with Hirschsprung's disease (N = 13), there were 12 positive reactions, and one false negative reaction in a neonate with total colonic aganglionosis. All biopsies from 24 unaffected children demonstrated negative reactions with no false positive reaction. In comparison, of the 35 specimens examined by H&E staining, ganglion cells were present in the submucosal Meissner's plexus only in 15 of these 24 unaffected children. In conclusion, a 97% diagnostic accuracy was achieved with AChE histochemistry compared with a 74% accuracy with H&E staining (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Preservation of acetylcholinesterase enzyme activity in non-frozen rectal biopsy specimens for Hirschsprung disease

Abstract

The acetylcholinesterase (AChE) stain has been a commonly accepted adjunctive study for rectal biopsy specimens in patients being evaluated for Hirschsprung disease (HD). However, the requirement for immediately frozen rectal biopsy specimens (to preserve the enzyme activity) has proved to be an obstacle to its use in some centers. In this study, we developed a microwave procedure for the AChE stain, which we then used to evaluate a special tissue transport medium, Michel’s medium, for its utility in preserving AChE enzyme activity without immediate freezing in fresh rectal specimens collected from known HD patients. When compared with similar rectal biopsy specimens that were immediately snap-frozen, the specimens stored in Michel’s medium at room temperature or with refrigeration (for up to 108 hours) followed by sucrose cryoprotection showed preservation of AChE enzyme activity, with comparable nerve fiber staining and no significant background staining. This technique provides a convenient alternative method to immediate snap-freezing for the demonstration of AChE enzyme activity and allows the courier transport or mailing of specimens without freezing to laboratories where this special stain is performed routinely.     

Modified H & E staining technique for fine needle aspiration cytology (FNAC) smears

Papanicolaou (Pap) staining procedure has achieved worldwide acceptance in cytology practice due to its crisp cytological details. There are many centres or private laboratories in our country which cannot fulfill the economic requirement of Pap staining and hence employ comparatively cheaper haematoxylin and eosin (H & E) stain. Although routine H & E cannot replace Pap, this study is an attempt to modify H & E staining that would offer comparable diagnostic results. The present study is restricted to FNAC material from palpable lesions i.e.breast and lymph nodes. For this purpose 50 lymph nodes ( LN) and 18 breasts were aspirated. Out of two fixed smears, 1 was stained by Pap technique for routine reporting and other by modified H & E method which was examined and reported by other pathologist, Dr.Sangeeta B.Desai ( SBD) The diagnosis of both the techniques were compared. Emphasis was also given on cytomorphological characteristics. Out of fifty lymph node aspirates from various sites, no diagnostic discrepancy was observed in 46 cases. Three out of 4 had sampling errors whereas, poor nuclear staining was noticed in a single case. Out of eighteen breast aspirates concordant diagnosis was achieved in 16 cases. Out of two discrepant diagnosis 1 was due to sampling error, and the other was an interpretative error. All the cases were confirmed histologically. In conclusion, modified H & E staining is useful for common sites of aspirations of superficial lesions.     

Biomarkers in Diagnosis of pancreatic carcinoma in fine-needle aspirates

This study was undertaken to determine whether recently identified proteins could be translated to clinical practice as markers to distinguish pancreatic adenocarcinoma from chronic pancreatitis on fine-needle aspirate (FNA) samples. Resected pancreatic tissue sections (n = 40) and FNA samples (n = 65) were stained for clusterin-beta, MUC4, survivin, and mesothelin. For each biomarker, the staining patterns in adenocarcinoma and in reactive ductal epithelium were evaluated and compared. Clusterin-beta stained reactive ductal epithelium significantly more frequently than pancreatic adenocarcinoma (P < .001). In comparison, MUC4 and mesothelin were expressed more frequently in pancreatic adenocarcinoma on tissue sections. Positive staining for MUC4 (91% vs 0%; P < .001) and mesothelin (62% vs 0%; P = .01) and absence of staining for clusterin-beta (90% vs 7%; P < .001) were noted significantly more frequently in adenocarcinoma cells than in reactive cells in FNA samples. Clusterin-beta and MUC4 can help distinguish reactive ductal epithelial cells from the cells of pancreatic adenocarcinoma in FNA samples.     

Evaluation of crystals in formalin-fixed, paraffin-embedded tissue sections for the differential diagnosis of pseudogout, gout, and tumoral calcinosis.

Hematoxylin-eosin (H&E)-stained sections may not allow proper evaluation of birefringence properties of the crystals in the lesions of pseudogout, gout, and tumoral calcinosis. This study was undertaken to verify the application of a special stain that could facilitate the evaluation of the birefringence properties of these crystals for definitive diagnosis. We evaluated previously described nonaqueous alcoholic eosin staining (NAES) method based on the principle of using alcoholic eosin without hematoxylin and any other aqueous reagents for staining of formalin-fixed, paraffin-embedded tissue sections. Two observers, in a blinded fashion, evaluated the sections stained with routine H&E and NEAS method without the knowledge about clinical diagnosis. All pseudogout (nine sections from seven cases) and gout (eight sections from five cases) lesions demonstrated birefringence in the sections stained with NAES method. H&E-stained sections showing the respective diagnostic histomorphology failed to demonstrate the birefringent crystals by polarizing microscopy in all the eight sections from gout and in seven of nine sections from pseudogout. Only two H&E-stained sections showed scant calcium pyrophosphate dihydrate (CPPD) crystals in pseudogout. None of the three sections from two cases of tumoral calcinosis showed birefringence with either stain. We conclude that CPPD in pseudogout and monosodium urate in gout may not polarize in the routine H&E-stained sections. However, polarizing microscopy of sections stained with NAES method allowed demonstration of CPPD crystals with positive birefringence in pseudogout, MSU crystals with negative birefringence in gout, and calcium hydroxyapatite crystals without birefringence in tumoral calcinosis. Section stained with NAES method is a significantly useful adjunct to the routine H&E stain for proper evaluation of the crystals under polarizing microscope in these lesions.     

Pseudoactinomycotic radiate granules of the gynaecological tract: review of a diagnostic pitfall

The filamentous bacterium actinomyces can cause serious gynaecological tract infections, including pelvic inflammatory disease (PID) and tubo-ovarian abscess. Thus, definitive diagnosis of actinomycotic granules (AMGs) in gynaecological specimens is clinically important. Non-infectious pseudoactinomycotic radiate granules (PAMRAGs) can mimic the microscopic appearance of AMGs. PAMRAGs may be more common than actinomycotic infections in specimens from patients using intrauterine devices and may be seen in patients with PID. Although the composition and aetiology of PAMRAGs is unclear and variable, a panel of histochemical stains can aid in diagnosis. On haematoxylin and eosin (H&E) stained sections, AMGs show as distinct granules with basophilic peripheral radiating filaments and a dense central eosinophilic core, whereas H&E stained sections of PAMRAGs feature refractile granules with irregular club-like peripheral projections and no central dense core. The filaments of AMGs are Gram positive on Brown and Brenn (B&B) stain and are highlighted with Gomori methenamine silver stain (GMS). They stain negatively with a modified acid fast bacillus (AFB) stain, aiding in the distinction of actinomyces from nocardia. PAMRAGs show negative or non-specific staining with B&B, GMS, and AFB stains. Therefore, knowledge of these staining properties and the distinguishing characteristics of PAMRAGs and AMGs enables recognition of this important diagnostic pitfall.     

Will modified Papanicolaou stain be the new stain for keratin?

The aim of the study is to compare hematoxylin and eosin (H&E) stained tissue sections with Papanicolaou-stained (PAP-stained) tissue sections. Paraffin block tissue sections of five oral pathologies were prepared and stained routinely with H&E and PAP stains. The results of the stained slides revealed a marked difference in staining with respect to color balance, contrast and intensity between H&E and PAP stains. Areas of keratinization were stained from shades of orange to pink depending on the degree of keratinization. In moderately differentiated squamous cell carcinoma, areas of keratin pearl formation and individual cell keratinzation were clearly demonstrated; these were not apparent in the H&E stained slides. The review of this study suggest that the PAP stain more clearly demonstrated key morphological features, associated with keratinization, and it may be considered as a supplemental staining procedure, aiding in diagnosis.     

Enumeration and immunohistochemical characterisation of bone marrow basophils in myeloproliferative disorders using the basophil specific monoclonal antibody 2D7

BACKGROUND: Basophils are highly specialised granulocytes that express a unique profile of antigens and increase in myeloproliferative disorders (MPD). In chronic myeloid leukaemia (CML), basophilia is a diagnostic and prognostic determinant. So far, however, no reliable approach for routine detection and enumeration of bone marrow basophils has become available. OBJECTIVE: To detect and enumerate basophils in bone marrow sections in patients with CML and other MPD. METHODS: The anti-basophil antibody 2D7 was applied to paraffin embedded bone marrow sections from normal/reactive subjects (n = 31), patients with CML (chronic phase, n = 37; accelerated phase, n = 9), and other MPD (chronic idiopathic myelofibrosis (CIMF), n = 20; polycythaemia vera (PV), n = 20; essential thrombocythaemia (ET), n = 20; indolent systemic mastocytosis (ISM), n = 7). RESULTS: As assessed by serial section staining, 2D7(+) cells were found to co-express myeloperoxidase, histidine decarboxylase, CD9, and CD43, but did not express B cell or T cell restricted antigens. 2D7(+) bone marrow cells were found to increase in CML compared with normal/reactive bone marrow and other MPD (median numbers of 2D7(+) cells/mm(2): CML, 33; normal/reactive bone marrow, 6; CIMF, 10; PV, 6; ET, 5; ISM, 3; p<0.05). The highest basophil counts were recorded in accelerated phase CML (115/mm(2)). CONCLUSIONS: A novel immunohistochemical procedure has been established for basophil detection in normal bone marrow and MPD. This approach should help in the quantification of bone marrow basophils at diagnosis and during anti-leukaemic treatment.     

Histological staining of pre- and postsynaptic components of amphibian neuromuscular junctions

Summary We have developed a combined staining technique whereby pre- and postsynaptic structures of the neuromuscular junction can be simultaneously visualized in the light microscope. The general approach was first to stain presynaptic nerve terminals using nitroblue tetrazolium (NBT), which when reduced to its diformazan state coloured the entire nerve terminal arborization blue. When the NBT stain was combined with the Karnovsky acetylcholinesterase (AChE) procedure the blue-coloured nerve terminal processes were vividly outlined by the brown copper ferrocyanide-AChE reaction product. Experiments were performed to ensure that the NBT-AChE method stained nerve reliably and that even extremely small neural processes were stained and visualized in the light microscope. This method stained cholinergic neuromuscular junctions and unmyelinated axons in a variety of preparations. Aldehyde pre- and poststaining fixation markedly affected the quality of NBT nerve stain. In addition, glutaraldehyde had a direct role in the staining process. The quality of the nerve terminal staining was affected by the pH and the constituents of the staining solution. The powerful experimental advantage of the NBT-AChE stain for neuromuscular junctions resulted from the sharp colour contrast which made possible accurate determinations of the relationship between the presynaptic nerve terminal arborization and the postsynaptic junctional AChE activity.     

Comparison of staining methods and a nested PCR assay to detect Histoplasma capsulatum in tissue sections

To optimize diagnosis of histoplasmosis in tissue sections, 30 spleen specimens from mice, experimentally infected with Histoplasma capsulatum, were examined by H&E, Grocott stain, anti-bacille Calmette-Guerin antibody immunostain, Fungiqual A fluorochrome stain (Drs Reinehr and Rembold, Kandern, Germany), and a nested polymerase chain reaction (PCR) assay. Results were compared with the tissue burden determined by quantitative culture. By applying logistic regression, the nested PCR assay was the most sensitive method, but not significantly more sensitive than the Grocott stain. The 50% quantile to achieve a positive result was determined to be 3 colony-forming units per milligram of spleen tissue for the PCR assay, 11 for the Grocott stain, 27 for the fluorochrome stain, 190 for immunostaining, and 533 for the H&E stain. The Grocott and fluorochrome stains did not differ significantly in detecting fungal elements. The PCR assay unambiguously identified H. capsulatum in tissue sections.     

Cellular immunolocalization of S100 protein within fixed tissue sections by monoclonal antibodies

Monoclonal antibodies (MoAb) that cross-react with the shared epitopes of S100 protein have been prepared from mouse hybridoma cell lines and partially characterized. Nine of these MoAb were applied to sections of formaldehyde-fixed paraffin-embedded human tissues that were stained by immunohistochemical techniques. Three of these MoAb give uniformly and reproducibly positive staining in appropriate cell types when stained by avidin-biotin methods. Three of the MoAb were judged to be negative, although some MoAb gave inappropriate staining patterns. The three remaining MoAb showed either great heterogeneity in their staining patterns or intensities, or gave a lesser degree of reproducibility in a given tissue or neoplasm. One of the MoAb designated 15E2E2 that belonged to the first group of reproducibly staining antibodies was used to stain a larger number of normal human tissues and neoplasms. The staining that was observed appeared to recapitulate that which was previously described for conventional S100 protein antibodies. Monoclonal antibodies may, therefore, have a role in selected cases where standard microscopy is equivocal for a specific tissue diagnosis, or where independent verification of the diagnosis would be beneficial.     

Conclusions from a study of venous invasion in stage IV colorectal adenocarcinoma

AIMS: Venous invasion is an established predictor of prognosis in colorectal cancer (CRC). The reported incidence of venous invasion in CRC specimens varies between 10% and 89.5%, mainly as a result of interobserver variability and differences in specimen processing (for example, staining with haematoxylin and eosin (H+E) alone versus the addition of an elastic fibre stain). This study was performed with three purposes in mind, namely: (1) To assess and compare the incidence of venous invasion diagnosed on H+E stained tissue versus tissue stained with both H+E and an elastic fibre stain. (2) To estimate the inherent false negative rate associated with the diagnosis of venous invasion by histopathological evaluation of resected CRC specimens. (3) To compare the resulting data regarding incidence, quantity, site, and type of venous invasion to the pertinent literature. METHODS: Venous invasion was assessed on sections from 81 CRCs resected from patients with synchronous distant metastases (hepatic and non-hepatic). Only stage IV tumours were studied for the following reasons: (1) it can be assumed that in all patients with distant haematogenous metastases venous invasion had occurred, thus enabling the false negative rate to be calculated; (2) there can be no dispute about the clinical relevance of the various characteristics of venous invasion identified in the tumours of patients with synchronous distant haematogenous metastases; and (3) to eliminate the effect of variance in tumour stage on the incidence of venous invasion. Initially, H+E stained sections were studied for venous invasion. Sections that were negative or questionable with regard to venous invasion were then stained with an elastic fibre stain, and a second search for venous invasion was carried out. Venous invasion was characterised by incidence, quantity, type, and site. The chi(2) test for independence was used to compare the incidence of venous invasion in colonic versus rectal and rectosigmoid primary tumours, and in patients with hepatic versus non-hepatic metastases. RESULTS: Venous invasion was identified in 42 (51.9%) (of the 81 specimens on H+E stained sections. The addition of the elastic fibre stain enabled the diagnosis of venous invasion in 15 (38.5%) of the remaining 39 specimens, increasing the overall incidence to 57 of 81 cases (70.4%). Of the 57 positive specimens, venous invasion was minimal in 27 (47.4%), intermediate in five, (8.8%) and massive in 25 (43.9%). Only intramural veins were involved in 18 (31.6%), only extramural veins in 26 (45.6%), and both intramural and extramural veins in 13 (22.8%) of the 57 positive specimens. The filling type of venous invasion was found in 41 (71.9%), the floating type in 28 (49.1%), and the infiltrating type in six (10.5%) of the 57 positive specimens. There was no significant difference between the incidence of venous invasion in the colon (42 of 60; 70%) versus rectal and rectosigmoid tumours (15 of 21; 71.4%; p = 0.8539), nor in the incidence of venous invasion in patients with hepatic (49 of 70; 70%) versus non-hepatic (eight of 11; 72.7%) metastases (p = 0.9018). CONCLUSIONS: The addition of an elastic fibre stain enables the identification of venous invasion in a considerable proportion of sections from CRC tumours that are falsely negative for venous invasion on H+E stain alone. The inherent chance of missing venous invasion on histopathological evaluation of CRC tumours stained with H+E and elastic fibre stains is at least 10.5%, and may be as high as 29.6%. In a large proportion of stage IV CRCs, despite the presence of synchronous distant metastases, only a minimal extent of venous invasion (that is, one to two involved veins) is demonstrable in the primary tumour. This suggests that only minimal venous invasion is required for the seeding of clinically relevant haematogenous metastases, and emphasises the careful, dedicated search for venous invasion that is required from the pathologist. Although extramural venous invasion was predominant in stage IV CRCs, in a considerable proportion of tumours (about a third) only intramural venous invasion was found. This suggests that intramural venous invasion may also seed clinically relevant haematogenous metastases, and should therefore also be considered as an indicator of poor prognosis.     

Staining methods for morphometric studies of parietal and gastrin cells in the rat stomach

In order to select the most suitable procedures for quantitative microscopy of both parietal and gastrin cell populations in the rat stomach, various staining methods were compared. For parietal cell identification in particular, the following procedures were tested: i) the modification of the haematoxylin-eosin method proposed by Marks and Drysdale, ii) the haematoxylin-eosin-saffron fluorochrome stain on paraffin sections, iii) the haematoxylin-azophloxin-saffron fluorochrome stain on paraffin sections, and iv) the May-Grunwald-Giemsa stain on thin sections from plastic-embedded specimens. This last provided the best results in parietal cell individualization and seemed to be the most suitable method for an accurate image analysis. Immunohistochemistry was the only unequivocal way to identify gastrin cells. Two variant procedures were examined; a) the agar-paraffin embedding technique, and b) the combination of a mucin staining with the immunoperoxidase reaction. The first technique provided an easier procedure for handling seriate strips of gastric mucosa for proper enumeration of immunostained cells. The second was presented as a promising variant procedure for a combined investigation of both G-cell population and mucin secretion patterns under differnt experimental conditions in the same specimen.     

eIF4E as a marker for cervical neoplasia.

Eukaryotic translation initiation factor 4E (eIF4E) is upregulated in cancers of the breast and head and neck. The authors have shown that eIF4E is increased in cervical neoplasia and that eIF4E upregulates human papillomavirus (HPV) oncoprotein E7. The aim of this study was to quantitate eIF4E in tissues representing a wide range of cervical pathology. The potential correlation between dysplastic grade or tumor stage with eIF4E upregulation and/or HPV genotype was analyzed for 10 normal, 27 cancer, and 37 dysplasia cases. A progressive increase in eIF4E staining intensity was found with increasing cervical pathology (P < 0.0001). No difference was seen in eIF4E stain intensity by either tumor type--squamous cell cancer (n = 18), adenocarcinoma (n = 4), or other types of cancer (n = 5) (P = 0.97)--or by tumor grade--II (n = 5) versus III (n = 7). Likewise, neither an HPV typing result of HPV 16 (n = 10) versus non-HPV 16 (n = 4) nor single HPV infection (n = 11) versus dual HPV infection (n = 3) significantly altered the eIF4E stain results (P = 0.86 and 0.97, respectively). These results indicate that eIF4E stain intensity may be useful as a marker for cervical neoplasia.     

A useful panel for the diagnosis of Hirschsprung disease in rectal biopsies: calretinin immunostaining and acetylcholinesterase histochesmistry

The pathological evaluation of rectal biopsies for the diagnosis of Hirschsprung disease has been a challenging issue. We analyzed prospectively the usefulness of calretinin immunostaining and acetylcholinesterase (AChE) histochesmistry in rectal biopsies for the diagnosis of Hirschsprung disease. Frozen tissue samples from 43 patients were used for AChE histochemistry and paraffin-embedded sections for calretinin immunohistochemistry and conventional histology (hematoxylin and eosin [H&E]). Activity for AChE, was demonstrated in 13 of 43 cases, and the absence of immunoreactivity for calretinin was observed in 14 of 43 cases. Conventional histology (H&E) did not reveal ganglion cells in 24 of 43 cases. The results on calretinin were in good agreement with AChE according to the κ index (0.946; P < .001) and presented significantly higher specificity (96.7 × 63.3; P < .002) and accuracy (97.6 × 74.4; P < .003) when compared with conventional histology (H&E). The final diagnosis of Hirschsprung disease was confirmed in 13 of 43 patients who were submitted to surgical treatment. The results of the present study indicate that calretinin can be a good tool in ruling out the diagnosis of Hirschsprung disease, by showing positive staining in ganglion cells and intrinsic nerve fibers, whereas AChE is useful in confirming the diagnosis of Hirschsprung disease, by revealing activity of this enzyme in hypertrophied nerve fibers. The association between calretinin and AChE can be a useful panel for the histopathologic evaluation of rectal biopsies for the diagnosis of Hirschsprung disease.     

Hep par 1 antibody stain for the differential diagnosis of hepatocellular carcinoma: 676 tumors tested using tissue microarrays and conventional tissue sections.

A well-characterized positive marker for hepatocellular differentiation would be a useful tool for the diagnosis of hepatocellular carcinoma (HCC). The recently commercially available Hep Par 1 antibody (clone OCH1E5.2.10) has been reported to be a sensitive marker for HCC in paraffin embedded sections. Of non-hepatocellular tumors, occasional carcinomas have been reported to stain, most frequently gastric adenocarcinomas. This study further evaluated the staining of this antibody on a large number of neoplasms using tissue microarray technology as well as conventional tissue sections. Six hundred seventy-six tumors, including 19 cases of HCC, were tested. Eighteen of 19 cases of HCC were positive, 3 showing <5% staining. Two cases negative on the array showed focal staining when whole tissue sections from the same tumors were used. 16 of 34 cases of gastric carcinomas gave positive reactions, 4 of these showed less than 5% staining. Staining of gastric carcinomas was not limited to signet ring-type carcinomas or to areas of hepatoid differentiation. Only 1 of 11 cases of cholangiocarcinoma showed focal staining. We also noted several other tumors to stain occasionally, including adrenal cortical carcinoma (3/13), yolk sac tumor (2/9), colonic adenocarcinoma (8/106), lung carcinoma (3/52), ovarian carcinoma (5/48), and endocervical adenocarcinoma (1/5). We did not observe staining in pancreatic carcinoma (11), renal cell carcinoma (36), breast carcinoma (85), melanoma (25), or mesothelioma (5). This study supports Hep Par 1 as a useful marker in the differential diagnosis of HCC, but with significant limitations. Cautious use of this antibody in a panel with other positive (alpha fetoprotein, CD10, polyclonal carcinoembryonic antigen) and negative (epithelial membrane antigen, monoclonal carcinoembryonic antigen, CD15) markers of hepatocellular differentiation may aid in the accurate diagnosis of HCC.     

Digital simulation of staining in histopathology multispectral images: enhancement and linear transformation of spectral transmittance

Hematoxylin and eosin (H&E) stain is currently the most popular for routine histopathology staining. Special and/or immuno-histochemical (IHC) staining is often requested to further corroborate the initial diagnosis on H&E stained tissue sections. Digital simulation of staining (or digital staining) can be a very valuable tool to produce the desired stained images from the H&E stained tissue sections instantaneously. We present an approach to digital staining of histopathology multispectral images by combining the effects of spectral enhancement and spectral transformation. Spectral enhancement is accomplished by shifting the N-band original spectrum of the multispectral pixel with the weighted difference between the pixel's original and estimated spectrum; the spectrum is estimated using M < N principal component (PC) vectors. The pixel's enhanced spectrum is transformed to the spectral configuration associated to its reaction to a specific stain by utilizing an N × N transformation matrix, which is derived through application of least mean squares method to the enhanced and target spectral transmittance samples of the different tissue components found in the image. Results of our experiments on the digital conversion of an H&E stained multispectral image to its Masson's trichrome stained equivalent show the viability of the method.     

The diagnostic value of acetylcholinesterase/butyrylcholinesterase ratio in Hirschsprung's disease

Aganglionosis of large bowel (Hirschsprung's disease; HD) is associated with higher acetylcholinesterase activity (AChE activity). Occasionally, especially in the neonatal period, the AChE activity may not be of diagnostic value. The authors previously reported that simultaneous estimation of butyrylcholinesterase activity (BChE activity) and the determination of AChE/BChE ratio may have discriminatory diagnostic value. They extended this finding to 31 cases of HD, in 16 of which resected tissue was available for study. All cases had histologic confirmation of aganglionosis. The AChE/BChE ratio was found to be higher than 2.0, with the exception of a case in which the biopsy weight was low (i.e., less than 3 mg), even when the AChE activity was normal or borderline. The estimation of AChE/BChE ratio is easy, rapid, and, in the author's experience, of discriminatory diagnostic value.     

Topography of enkephalin, substance P and acetylcholinesterase staining in Huntington's disease striatum

The distribution of Met-enkephalin (Enk) and substance P (SP) was examined in the striatum of Huntington's disease (HD) patients using immunoperoxidase techniques. Both Enk- and SP-like immunoreactivities (ir) were strikingly diminished in the dorsal caudate nucleus and putamen, while patchy staining persisted in the ventral putamen and nucleus accumbens. This was in sharp contrast to the patch-matrix pattern of acetylcholinesterase (AChE) staining which persisted throughout the entire striatum in HD. The regional loss of Enk- and SP-ir parallels the pattern of neuronal depletion in HD. The disparity between AChE staining and Enk- and SP-ir in HD suggests that AChE-positive neurons or fibers are resistant to the destructive process in areas where intrinsic neuronal populations are depleted.     

Utilization of antihepatocyte clone OCH1E5 (Hep Par 1) in histological evaluation of liver tumors

Diagnosis of hepatocellular carcinoma (HCC) is not always easy on simple hematoxylin and eosin (H&E) stain. The diagnostic problems arise when tumor shows pseudoglandular, pleomorphic or clear cell differentiation. Various tumors markers have been described with varying sensitivity and specificity. Monoclonal antibody Hep Par 1 (OCH1E5) which is specific for hepatocytes offers great help in separation of these tumors. The aim of the present study was to determine utility of Hep Par 1 (OCH1E5) in differentiating HCC from metastatic tumors and cholangiocarcinoma. Total of 62 cases of liver tumors obtained from biopsies, resected or autopsy specimens were included in the study. Slides having representative sections were subjected to immunohistochemistry with monoclonal antibody Hep Par 1 (Dako Corp) using avidin biotin technique with primary antibody dilution of 1:40. Adjacent nontumorous hepatocytes were taken as positive control. Slides were examined by experienced pathologist without any information of clinical or H&E diagnosis. Cases were considered positive for Hep Par 1 if tumor cells showed cytoplasmic brown colored granules. The intensity and distribution (diffuse/ focal) of immunoreactivity was noted. Subsequently immunohistochemistry results were correlated with histology and clinical diagnosis. Hep Par 1 antibody was positive in 26 (42 %) and negative in 36 (58 %) liver tumors. On correlating with H&E sections, out of 26 positive cases, 25 (89.2%) were HCC and one was the case of metastasis of mucin secreting adenocarcinoma. From 36 tumors with negative staining 3 were cases of HCC, 27 metastatic adenocarcinomas and 6 cholangiocarcinomas. Only one case of liver metastasis of mucin secreting adenocarcinoma showed positivity. None of the cases of cholangiocarcinoma showed positivity for Hep Par 1. The three HCCs which did not take up staining for Hep Par 1 were 2 cases of moderately differentiated HCC having pseudoglandular pattern and a case of well differentiated HCC with trabecular arrangement. In 11(44%) cases staining was diffuse while in 14 (56%) it was focal but intense. Hep Par 1 is a useful marker in differentiating HCC from metastaic tumors and cholangiocarcinoma with sensitivity and specificity of 89 % and 97 % respectively and positive predictive value of 96 %. However one should be aware of limitations of immunohistochemistry.