Application of the membrane filter technique to bromodeoxyuridine immunochemistry for exfoliative cytology

The membrane filter technique for smear specimens of tumors in bromodeoxyuridine (BrdU) immunochemistry is described. The staining results of Raji cells processed using the filter technique was compared with that obtained by the conventional cytospin method. Although the BrdU mean labeling index (LI) for in cytospin specimens was almost the same as the LI in membrane filter specimens, filter specimens showed excellent staining and less cell destruction compared with those processed by cytospin. Small amounts of tumor specimens such as squamous cell carcinoma and polymorphous low-grade adenocarcinoma also were processed using the membrane filter appliance. For squamous cell carcinoma, the LI for the filter specimens was 5.36 ± 0.38 and that of the paraffin sections was 5.56 ± 0.38. The membrane filter technique provided relatively undamaged specimens for exfoliative cytology and will be useful for immunohistochemical evaluation of tumor cells and for routine, noninvasive cytological screening.     

Cytopathology of the central nervous system. Part I. Utility of crush smear cytology in intraoperative diagnosis of central nervous system lesions

OBJECTIVE: To evaluate the utility of rapid intraoperative crush smear cytologic diagnosis of central and peripheral nervous system lesions and to determine the accuracy and relevance of the accuracy of the intraoperative cytologic diagnosis when compared to the final paraffin section diagnosis. STUDY DESIGN: The crush (squash) smear technique was introduced at Sher-i-Kashmir Institute of Medical Sciences in May 2003. The 8 months of 2003 were used for standardization of the procedure. In 2004, 151 patients with open neurosurgical specimens or stereotactic biopsies were diagnosed intraoperatively by crush smears, and the diagnosis was compared with final diagnosis on paraffin sections of the same tissue samples. No supplementation of frozen sections was used. RESULTS: Of 151 cases, 144 were diagnosed accurately intraoperatively by crush smear cytology when compared with the respective paraffin section diagnoses. The diagnostic accuracy attained was 95.36%. Each case was diagnosed within 10 minutes after receipt of sample. Neurosurgical procedure (open or stereotaxy) did not affect diagnostic accuracy. CONCLUSION: In the expert hands of a pathologist with good exposure neurosurgical specimens, crush smear cytology is an accura and reliable procedure for the intraoperative diagnosis central nervous system tumors.     

Making Mock-FNA Smears from Fresh Surgical Pathology Specimens to Improve Smear Preparation Technique and to Create Cytohistological Correlation Series

Background

Fine needle aspiration (FNA) cytology is a well-established diagnostic method based on the microscopic interpretation of often scant cytological material; therefore, experience, good technique and smear quality are equally important in obtaining satisfactory results.

Aims of Study

We studied the use of fresh surgical pathology specimens for making so-called mock-FNA smears with the potential of cytohistological correlation. Additionally, we studied how this process aids the improvement of preparation technique and smear quality.

Methods

Cytological aspirates from 32 fresh biopsy specimens from various sites: lung (20), lymph nodes (6), and breast (6) were obtained, all with a clinical diagnosis of tumor. Aspiration was performed from grossly palpable tumors. 25G needle and Cameco-type syringe holder was used with minimal or no suction.

Results

Unfixed surgical specimens provided sufficient cytological material that resulted in good quality smears. After standard processing of specimens into microscopic sections from paraffin embedded tissues, cytohistological case-series were created. No significant alteration was reported in tissue architecture on hematoxylin-eosin stained sections after the aspiration procedure. A gradual, but steady improvement was observed in smear quality just after a few preparations.

Discussions and Conclusions

Our study proved that surgical specimens may be used as a source of cytological material to create cytohistological correlation studies and also to improve FNA cytology skills. The use of very fine gauge needle (25G, 0,6 mm diameter) during the sampling process does not alter tissue architecture therefore the final histopathological diagnosis is not compromised. We conclude that by using fresh surgical specimens useful cytohistological collections can be created both as a teaching resource and as improving experience.     

Serial Sectioning of Insects with Hard Exoskeleton by Dissolution of the Exocuticle

The method reported here was designed to produce paraffin serial sections as thin as 5 Mm of insects or other arthropods with a hard cuticle. Heads and abdomens of Apis mellifera, Eristalomyia tenax and Tenebrio molitor were fixed with Schaffer's liquid, dehydrated with 80% ethanol, 90% ethanol, two changes of 100% isopropanol (2 hr each) and 12 hr in a 1:1 mixture of paraffin (58 C melting point) at 60 C. They were molded in paraffin after 12 hr of infiltration under a partial vacuum at 60 C. Large body openings of objects were sealed with paraffin to prevent infiltration of solvents.

Thereafter, the outer paraffin was removed manually and with xylene (15 min); the cuticle was rehydrated with 100% isopropanol and 100% ethanol (15 min each). The objects were then treated with Sputofluol (Merck; a mixture of NaOH and NaCIO) until they became white or their colorless endocuticle was stainable with aniline blue WS (C.I. 42755) after rinsing in a 50% acetic acid solution (v/v). They were then dehydrated with 100% ethanol and 100% isopropanol (15 min each) and subsequently re-embedded in paraffin. They were molded, sectioned, stained and mounted as usual.     

Bromodeoxyuridine (BrdU) immunocytochemistry by exonuclease III (Exo III) digestion.

A new procedure is described to generate single-stranded DNA by exonuclease III (Exo III) digestion for bromodeoxyuridine (BrdU) immunocytochemistry on tissue sections. We compared this procedure with the most widely used procedure of DNA denaturation with 2 N HCl. In vivo and in vitro pulse and continuous labelling of tissues and cells were used. The specimens were fixed in formalin, ethanol, glutaraldehyde, Carnoy's, Bouin's or Zamboni's fixative and embedded in paraffin or used unfixed as cryostat sections or cytospin preparations. After Exo III digestion, BrdU substituted DNA was detected irrespective of the fixation procedure applied. The optimal protocol for nuclease digestion appeared to be simultaneous incubation, of 10 Units Exo III per ml EcoRI buffer and anti-BrdU monoclonal antibody at 37 degrees C. The advantages of Exo III digestion for BrdU immunocytochemistry compared to acid denaturation were: less non-specific nuclear background reactivity, no DNA renaturation, less DNA loss, optimal nuclear morphology, increase in antibody efficiency and the possibility for simultaneous detection of acid-sensitive tissue constituents. Disadvantages of the Exo III digestion are decreased sensitivity and the need for more rigorous pepsin pretreatment. We conclude that Exo III digestion of DNA is an appropriate alternative for acid denaturation for BrdU immunocytochemistry on sections of pulse-labelled specimens.     

Orthochromatic, Species-Limited Staining of Mast Cells With Night Blue

Night blue will stain the mast cells of rat, mouse and hamster selectively if alcohol differentiation is controlled. The technical steps are: Dewax paraffin sections with xylene, 2 changes; air dry; 2% Na₂SO₄, 3-5 sec; 0.5% night blue in 10% ethanol, 1 hr at 60°C; rinse in water; 9% HNO₃, 15 sec; water 1-5 min; 70% ethanol, 2 changes, 30 sec each; wash; 0.01% safranin, 3-5 sec; rinse, blot, air dry, mount in synthetic resin. A clear orthochromatic stain of the mast-cell granules occurs. Acid fixation prevents the staining reaction.     

Methods of cytologic smear preparation and fixation. Effect on the immunoreactivity of commonly used anticytokeratin antibody AE1/AE3

OBJECTIVE: To evaluate the effect of fixation and methods of cytologic smear preparation on the immunoreactivity of commonly used anticytokeratin antibody AE1/AE3. STUDY DESIGN: Scrape cytology smears and formalin-fixed, paraffin-embedded tissue sections (FPTS) of 20 unfixed, fresh specimens submitted for intraoperative consultation were studied by the immunoperoxidase method. In addition to the morphologic examination, the smears and FPTS were evaluated for intensity and proportion scores. For each specimen, two scrape cytology smears were wet fixed in 95% ethanol, and 12 smears were air dried without fixation. Air-dried smears were either postfixed after rehydration in saline or fixed directly without rehydration by one of the three fixatives: alcoholic formalin, 95% ethanol with 5% acetic acid or 95% ethanol. RESULTS: Both intensity and proportion scores were higher with rehydrated, air-dried smears as compared to those without rehydration and were comparable to those with wet-fixed smears and FPTS. In the rehydrated group, the optimum results were achieved when the smears were postfixed with alcoholic formalin. CONCLUSION: The method of preparation and fixation had variable effects on the immunoreactivity of anticytokeratin antibody AE1/AE3. The optimum results were achieved with saline-rehydrated, air-dried smears post-fixed in alcoholic formalin. To evaluate the role of inter-sample variation, further, larger studies are recommended on this and other antibodies before applying them to different types of cytologic smears.     

Complete penetration of antibodies into vibratome sections after glutaraldehyde fixation and ethanol treatment: light and electron microscopy for neuropeptides.

To develop a method for quantitative electron microscopic immunocytochemistry on neural tissue of CNS, we tested the extent to which ethanol treatment would improve the penetration of immunoreagents through vibratome sections fixed in high concentrations of glutaraldehyde without compromising ultrastructure. Transverse or sagittal vibratome sections (60-80 microns) of spinal cord perfused with 1% formaldehyde plus 1% or 2.5% glutaraldehyde were washed in 50% ethanol for 0-70 min and stained to reveal immunoreactivity for neuropeptide Y (NPY). Semi-thin (1 micron) or ultra-thin sections were used to assess the depth to which NPY nerve fibers in the dorsal horn were stained. Without ethanol washing, immunoreactive nerve fibers were visualized only in the surface 5-10 microns of transverse or sagittal vibratome sections. In transverse vibratome sections, NPY nerve fibers, which ran perpendicular to the cut surfaces of the sections, were entirely stained after a 30-min wash in 50% ethanol. The numbers of NPY-immunoreactive varicosities and synapses were comparable at the surfaces and in the centers of the vibratome sections. In sagittal sections, where NPY nerve fibers ran parallel to the cut surfaces, fibers in the centers of vibratome sections could not be labeled even after 70 min in 50% ethanol. Substance P- and enkephalin (Enk)-immunoreactive nerve fibers could also be completely stained in transverse sections of spinal cord or medulla oblongata after 30-min exposure to ethanol. Ethanol washing had no significant deleterious effects on ultrastructure, although the amount of cytoplasmic matrix in neurons decreased with increasing exposure. These results indicate that washing with 50% ethanol for at least 30 min allows immunoreagents to penetrate completely through nerve fibers fixed with high concentrations of glutaraldehyde, as long as the fibers have cut ends at both surfaces of a vibratome section. This technique makes possible quantitative electron microscopic immunocytochemical studies and is proving a useful tool for defining synaptic connections in the CNS.     

Thick Sections for Embryology

Night blue will stain the mast cells of rat, mouse and hamster selectively if alcohol differentiation is controlled. The technical steps are: Dewax paraffin sections with xylene, 2 changes; air dry; 2% Na₂SO₄, 3-5 sec; 0.5% night blue in 10% ethanol, 1 hr at 60°C; rinse in water; 9% HNO₃, 15 sec; water 1-5 min; 70% ethanol, 2 changes, 30 sec each; wash; 0.01% safranin, 3-5 sec; rinse, blot, air dry, mount in synthetic resin. A clear orthochromatic stain of the mast-cell granules occurs. Acid fixation prevents the staining reaction.     

建立小鼠膀胱肿瘤原位模型的优化方法

目的探讨硝酸银、盐酸、胰酶和乙醇预处理构建鼠膀胱肿瘤的成瘤机制。方法 24G静脉留置针插入膀胱,PBS冲洗后,将小鼠随机分为5组,每组6只:(1)乙醇作用组:22%乙醇0.1 mL保留20 min;(2)胰酶作用组:0.2%胰酶保留30 min;(3)酸碱作用组:0.1 mmol/L HCl 0.1 mL作用15 s后,PBS冲洗,0.1 mmol/L NaOH0.1 mL作用5 s,排空膀胱;(4)硝酸银作用组:0.15 mol/L硝酸银保留10 s;(5)对照组:0.1 mL生理盐水。术后1和24h随机处死每组3只小鼠,摘取膀胱,HE染色观察膀胱黏膜病理变化;戊二醛固定,电镜下观察膀胱黏膜细胞微结构变化;甲苯胺蓝染色,观察膀胱黏膜固有层肥大细胞数目变化;过碘酸-希夫(PAS)染色,观察膀胱黏膜GAG层变化。40只小鼠应用上述前四组预处理因素处理膀胱后,建立膀胱癌原位模型,计算各组成瘤率。结果胰酶和乙醇处理1h后,局部上皮伞状细胞脱落,黏膜下层暴露;酸碱和硝酸银处理组大部黏膜完整性破坏,黏膜下层暴露较多,连续性中断;对照组和实验组间炎症细胞浸润均不表现出统计学差异。24 h后,胰酶和乙醇组可见局部轻度水肿并充血,黏膜完整性恢复较好,细胞间见紧密连接;而酸碱和硝酸银组上皮黏膜薄厚不均一,仍可见部分脱落黏膜。结论利用硝酸银和酸碱预处理膀胱可作为鼠膀胱肿瘤原位模型构建的首选方法。     

Histochemical Application of Mild Alkaline Hydrolysis for Selective Elimination of O-Glycosidically Linked Glycoproteins

A new technique to eliminate O-glycosidically linked glycoprotein (mucin-type glycoprotein) selectively has been developed. Composite paraffin sections were collodionized before and after alkaline treatment with 0.5 M NaOH in 70% ethanol; the effect of this procedure on mucosubstances was examined using the periodic acid-Schiff reaction. Exposure to alkaline hydrolysis for 72 to 144 hours at 4 C led to a complete loss of periodic acid-Schiff reactivity of epithelial mucins in rat sublingual gland, stomach and small intestine, but that of fuzzy coat, thyroid colloid, collagen fibers and tracheal cartilage was well preserved. These results agreed fairly well with biochemical findings. The present study also revealed that materials prepared by freeze-substitution provided the most satisfactory results.     

Histochemical application of mild alkaline hydrolysis for selective elimination of O-glycosidically linked glycoproteins

A new technique to eliminate O-glycosidically linked glycoprotein (mucin-type glycoprotein) selectively has been developed. Composite paraffin sections were collodionized before and after alkaline treatment with 0.5 M NaOH in 70% ethanol; the effect of this procedure on mucosubstances was examined using the periodic acid-Schiff reaction. Exposure to alkaline hydrolysis for 72 to 144 hours at 4 C led to a complete loss of periodic acid-Schiff reactivity of epithelial mucins in rat sublingual gland, stomach and small intestine, but that of fuzzy coat, thyroid colloid, collagen fibers and tracheal cartilage was well preserved. These results agreed fairly well with biochemical findings. The present study also revealed that materials prepared by freeze-substitution provided the most satisfactory results.     

Quantitative histochemical investigations of semipermeable membrane techniques for the assay of acid phosphatase in skeletal muscle

Summary Meijer's semipermeable membrane technique for acid phosphatase was modified by treating sections immediately after incubation with 70% ethanol for 30 min at room temperature instead of with formalin. The modification improved the validity of the technique considerably, by stabilising the specific final reaction product formed in presumed enzyme-containing sites. The modified technique thus seems promising for assaying the activity of acid phosphatase in sections of skeletal muscle.     

Immunocytochemistry on fine needle aspirates in paraffin miniblocks

A simplified method of processing of fine needle aspirates for paraffin miniblocks suitable for both morphologic and immunocytochemical evaluation is described. Aspirates were fixed in ethanol at 4 degrees C, dehydrated in acetone and xylene and embedded in paraffin (58 degrees C). All steps were carried out in a single Eppendorf centrifuge tube; the total process took less than four hours. Deparaffinized sections were stained using the alkaline phosphatase-antialkaline phosphatase technique with monoclonal and conventional antibodies helpful in the differential cytologic diagnosis of alcohol-fixed aspiration biopsy specimens. Antibodies to keratin, vimentin, desmin, neurofilaments, glial fibrillary acidic protein, leukocyte-common antigen, synaptophysin and immunoglobulin kappa and lambda light chains reacted positively on the miniblock material. Since the paraffin miniblocks combine the histologic pattern of the tumor with the differentiation-specific information provided by immunocytochemistry, their use can improve the accuracy of tumor typing in aspirates.     

In vitro immunohistochemical localization of S-phase cells by a monoclonal antibody to bromodeoxyuridine

Thin viable slices of normal or pathological human tissues were incubated in vitro with bromodeoxyuridine (BrdU). Later, cryostatic sections and histological sections from the same samples embedded in paraffin were examined by an immunohistochemical method using a monoclonal antibody anti-bromodeoxyuridine (anti-BrdU-MAb): on both cryostatic and histological sections, the nuclei of the S-phase cells proved positive. The optimization of the technique depends on the concentration of bromodeoxyuridine in the culture medium (160 microM), the duration of incubation (not less than two h), the method of DNA denaturation (2N or 4N HCl) and the dilution of the anti-BrdU-MAb (1:50). In vitro, immunohistochemical application of the BrdU/anti-BrdU-MAb method permits a quantitative assessment of the proliferative activity of a tissue as well as the direct location of the actively replicating cells in histological sections.     

A Durable Nissl Stain for Frozen and Paraffin Sections

Frozen sections, 25-50 /j. thick, of formalin-fixed nervous tissues are mounted following the Albrecht gelatin technic. Paraffin sections, 15 p., are deparaffinized and transferred to absolute ethanol. The slides are then coated with celloidin. Both frozen and paraffin sections subsequently follow the same steps: absolute ethanol-chloroform (equal parts) for at least 20 min, 95% ethanol, 70% ethanol (1-3 min), then rinsed in distilled water. Sections are stained in Cresylechtviolett (Chroma) 0.5% aqueous solution containing 4 drops of glacial acetic acid per 100 ml, rinsed in distilled water, agitated in 70% ethanol until excess stain leaves the slide, and rinsed in 95% ethanol. Sections are then dehydrated in absolute ethanol, followed by butanol, cleared in xylene, and enclosed in permount.     

Bromodeoxyuridine (BrdU) immunocytochemistry by exonuclease III (Exo III) digestion

Summary A new procedure is described to generate single-stranded DNA by exonuclease III (Exo III) digestion for bromodeoxyuridine (BrdU) immunocytochemistry on tissue sections. We compared this procedure with the most widely used procedure of DNA denaturation with 2 N HCl. In vivo and in vitro pulse and continuous labelling of tissues and cells were used. The specimens were fixed in formalin, ethanol, glutaraldehyde, Carnoy's, Bouin's or Zamboni's fixative and embedded in paraffin or used unfixed as cryostat sections or cytospin preparations. After Exo III digestion, BrdU substituted DNA was detected irrespective of the fixation procedure applied. The optimal protocol for nuclease digestion appeared to be simultaneous incubation, of 10 Units Exo III per ml EcoRI buffer and anti-BrdU monoclonal antibody at 37° C. The advantages of Exo III digestion for BrdU immunocytochemistry compared to acid denaturation were: less non-specific nuclear background reactivity, no DNA renaturation, less DNA loss, optimal nuclear morphology, increase in antibody efficiency and the possibility for simultaneous detection of acid-sensitive tissue constituents. Disadvantages of the Exo III digestion are decreased sensitivity and the need for more rigorous pepsin pretreatment. We conclude that Exo III digestion of DNA is an appropriate alternative for acid denaturation for BrdU immunocytochemistry on sections of pulse-labelled specimens.     

Techniques for Preparing and Staining Phytophthora Oospores in Soybean Rootlets

Nylon mesh tissue carriers were constructed to hold soybean rootlets through fixing, dehydrating and embedding. Mesh pieces three centimeters square were doubled and sealed at each end by heat. Tissue samples were placed inside with an identifying piece of aluminum foil and the carrier sealed. Rootlets were fixed in Karpechenko's solution, dehydrated in an alcohol series and infiltrated with paraffin. They were embedded in paraffin after removal from the carrier, and sectioned on a microtome. Sections were mounted on glass slides and deparaffinized. A new stain was developed to differentiate oospores of Phytophthora megasperma var. sojae formed in these rootlets. The stain was prepared by dissolving 100 mg bromphenol blue in 50 ml of 95% ethanol and adding 3 g silver nitrate. Procedure: 5 sec in 95% ethanol, 30 min in silver stain, tap water rinse, 5 sec in 95% ethanol, 1 sec in saturated methylene blue in ethanol, immediate rinse in tap water, dehydration in absolute ethanol, rinse in tertiary butanol and xylene and mount. Previous clearing of the tissue was not required, and no air bubbles accumulated within the mesh carrier. This low cost, permeable carrier preserved the minute tissue specimens throughout processing, and the simple, progressive stain clearly differentiated oospores from surrounding tissue.     

A Department Devoted to Abstracts of Books and Papers from Other Journals Dealing With Stains and Microscopic Technic in General

TO enable staining of insoluble calcium salts with glyoxal bis(2-hydroxyanil) (GBHA), the original solution containing 2 ml of 0.4% GBHA in absolute ethanol, and 0.3 ml of aqueous 5% NaOH, and limited to staining only soluble calcium salts, was modified as follows: 1, 2 ml of 0.4% GBHA in absolute ethanol in 0.6 ml of 10% aqueous NaOH; 11, 0.1 gm GBHA in 2 ml of 3.4% NaOH in 75% ethanol. To prevent diffusion and loss of calcium, the tissues were processed by the freeze-substitution or freeze-dry method and sections stained without removing the paraffin. Modification I is effective only when 1 or 2 drops placed on the section are evaporated gradually to dryness, concentrating the GBHA and NaOH on the insoluble calcium salts. Modification II is effective when dried or poured on the the section and allowed to stain for 5 min. The stained slides are immersed for 15 min in 90% ethanol saturated with KCN and Na₂CO₃ for specificity to calcium; rinsed and counterstained in 95% ethanol containing 0.1% each of fast green FCF and methylene blue; rinsed and dehydrated in ethanol; deparaffinized and cleared in xylene; and mounted in neutral synthetic resin. Although the modified methods tested on models failed to stain reagent grade CaCO₃ and Ca₃(PO₄)₂ crystals completely, apatite in developing vertebrae and calcified plaques in soft tissues were stained intensely red. The distribution of gross deposits of insoluble calcium salt in tissue sections corresponded with that shown in adjacent sections by the alizarin red S, ferrocyanide, and von Kossa methods. The modified GBHA method revealed smaller quantities of insoluble as well as soluble calcium salts discretely within cells where the other methods failed; also, calcium in cytoplasm of hypertrophied cartilage cells of developing vertebrae, and in cytoplasm of renal tubular cells of magnesium-deficient rats, not described previously, was demonstrated.     

Diagnosis of Helicobacter pylori in gastric brush and biopsy specimens stained by Rowmanowsky and immunocytochemical methods: comparison with the CLOtest

Helicobacter pylori has been implicated in the pathogenesis of chronic gastritis, gastric and duodenal ulcer, and possibly gastric carcinoma. The organism may be detected by invasive or non‐invasive methods with variable sensitivity. Paired gastric biopsy and gastric brush specimens were collected from 83 patients presenting with non‐ulcer dyspepsia. One biopsy was tested for urease using the CLOtest, the other was processed to paraffin and consecutive sections were stained with haematoxylin and eosin, modified Giemsa and anti‐ H. pylori antisera. The brush specimens were stained with a rapid Romanowsky stain (Hema‐Gurr) and anti‐ H. pylori . The CLOtest was positive in 31 cases, the Giemsa biopsy in 25, the anti‐ H. pylori biopsy in 27, the Hema‐Gurr smear in 27 and the anti‐ H. pylori smear in 19. The sensitivities of the methods after omitting one inadequate biopsy were 96%, 93%, 100%, 96% and 78%, respectively. The specificities were 93% for the CLOtest and 100% for the other methods. While immunocytochemical staining of gastric biopsies may be the most sensitive method for H. pylori identification, the cost and turn around time of the technique may preclude its routine use. Gastric brush cytology is a highly sensitive and specific method for H. pylori detection that is quick and simple to perform. Its application is recommended for the routine diagnosis of H. pylori infection.