The influence of fixatives and other variations in tissue processing on nuclear morphometric features

The influence on the area and numerical density of nuclei was investigated in 5-mm-thick slices of guinea pig liver for different fixatives and variations in tissue processing: delay in fixation, air drying, degree of acidity of 10% formalin (= 4% formaldehyde), Bouin and mercury-formalin fixatives, acetone and ethanol dehydration and understretching and overstretching of the paraffin-embedded sections. Air drying (either forced or as a result of delayed fixation), the type of fixative and the degree of acidity affected the nuclear area. Regarding the latter, nuclear area was approximately 25% lower for pH less than or equal to 3 as compared with pH greater than 5. In comparison with the standard tissue processing used, the nuclear density was higher after all of the variations studied (air drying, acetone dehydration and fixation). These findings indicate that nuclear area, in contrast to other tissue components, is relatively insensitive to variations in tissue processing. However, it is essential to regularly measure the pH of the fixative: deviations from pH = 7 should be carefully avoided in order to keep nuclear area variations as a result of tissue processing within acceptable limits.     

Application of morphometry in tumor pathology

The application of morphometry in tumor pathology is discussed, e.g., its use in studying the biology of tumors, in creating tumor classification(s), in creating methods for the identification of a tumor in the diagnostic context, and in characterizing diagnostic histopathology in absolute terms. In traditional subjective diagnostic histopathology, reproducibility can be defined satisfactorily, but the definition of accuracy is ambiguous; in morphometric histopathology, a satisfactory definition is found for both concepts but it may be difficult to separate them in practice. Morphometric histopathology can study parameters measured from sections or parameters derived from the primary measurements through calculations. In the histopathology of tumors, the following parameters have turned out to be specially valuable: densitometric measurements of nuclei, nuclear area, perimeter and form factors, nucleolar parameters, the number of mitotic cells per area, the cellularity, the volume fraction of the epithelium, and parameters associated with the fraction of tumor tissue in the sample. The standard deviation or other moments of the distribution of these measurements can be more relevant than the mean values of the results. This indicates that more attention should be given to sampling rules, which are important in defining the efficiency of the methods. For rational application of morphometric methods, it is very important to make a distinction between group morphometry and diagnostic morphometry. The latter engenders numerous sources of variation (variation in section thickness, variation in tissue processing, variation in the techniques of measurement, interobserver variation, interlaboratory variation, variation due to subjective interpretation, etc.), which are usually better controlled in group morphometry. The influence on morphometric parameters of variation in section thickness and tissue shrinkage during processing are discussed.     

Lipid droplets in the adrenal cortex of the rat. Preservation after tannic acid—paraformaldehyde—glutaraldehyde fixation and extraction during staining

Adrenocortical tissue from the rat was fixed in glutaraldehyde-paraformaldehyde-tannic acid with or without potassium pyroantimonate. An electron opacity was observed in lipid droplets from unstained sections of tissue with or without antimonate in the fixative and is most likely attributable to inclusion of tannic acid in the fixative. The opacity was largely removed after staining with uranyl acetate in absolute methanol followed by lead citrate. Removal of the opacity is attributable to staining in lead citrate, not uranyl acetate, because highly basic solution without lead also removes the density. An electron-opaque rim is present at the interface of lipid droplet and cytoplasm, although no distinct membranous structure is observable. The rim may correspond to myelin-like structures seen sometimes in lipid droplets from adrenocortical cells fixed by routine procedures employing pre-fixation with glutaraldehyde and post-fixation with osmium tetroxide. Results of this study point to the conclusion that ultrathin sections should be examined unstained in the validation of a new regime for processing tissues in electron microscopy.     

The influence of fixation procedure, embedding medium and section thickness on morphometric data in thyroid gland.

In this study, the effects of fixation procedures, embedding medium and section thickness on stereological measurements of normal thyroid were analysed. The following conclusions were drawn: A) the use of a single section for the analysis of a lobe is sufficient if this section is located in the central part of the lobe. B) fixation and embedding with glutaraldehyde-Epon leads to a larger shrinkage than Bouin-paraplast, but the difference between the two procedures is not significant. C) osmium post-fixation reduces the shrinkage induced by glutaraldehyde and lowers the axial deformation produced by sectioning. D) Bouin's fixative and paraplast embedding induce considerable shrinkage of the interstitial tissue. The shrinkage obtained with glutaraldehyde-Epon is less. However, it is still not known whether this difference is due to the fixative, or to the embedding procedure or to both. E) only in glutaraldehyde and osmium-fixed material, embedded in Epon, can follicles and colloids be assumed to be spherical in shape without significant errors.     

Optimizing specimen processing for ancient soft tissue specimens

Abstract

Despite many reports concerning processing of ancient soft tissues, scant attention has been paid to optimizing procedures for processing soft tissues that have been altered by taphonomic processes. To determine the best procedures, we investigated the rehydration solution, time of exposure to the solutions, fixative solution and exposure to heat. Processes were evaluated based on the minimum section thickness, degree of tissue fragmentation, definition of tissue architecture and penetration of stains. We found that in desiccated samples, tissue architecture was optimized by using Ruffer's solution for rehydration and Schaffer's solution as fixative, because these tissues require water restoration within the tissues due to their compacted character. Heating enhanced penetration of dyes in these specimens, which improved diagnosis. Saponified tissues that had suffered extensive decomposition were more labile and required slow water uptake. The best histological sections were obtained using Sandison's solution followed by fixation with formaldehyde and avoiding heat. To obtain the best results with paleohistological specimens, the procedure must be determined by the condition of the sample and by accounting for the nature of its damage.     

On the preparation of cryosections for immunocytochemistry

The key preparation steps in the Tokuyasu thawed frozen section technique for immunocytochemistry, namely freezing, sectioning, thawing, and drying, were studied. A spherical tissue culture cell was used as a model system. The frozen hydrated section technique indicated that glutaraldehyde-fixed, 2.1 M sucrose-infused pellets of cells were routinely vitrified by immersion in liquid nitrogen but water was crystallized when lower sucrose concentrations (0.6-1 M) were used. Quantitative mass measurements showed that the fixed cells are freely permeable to sucrose. The frozen hydrated sections were severely compressed but cell profiles regained their circular appearance upon thawing. The average section thickness of our frozen-hydrated sections was 110 nm: this was reduced to 30-50 nm upon thawing, washing, and air-drying. This change was accompanied by severe drying artifacts. By using the methyl cellulose drying technique, this collapse upon air-drying could be significantly reduced, but not completely prevented, giving an average thickness of 70 nm.     

包埋前原位TUNEL标记凋亡淋巴细胞的电镜观察

目的用包埋前原位尾端标记技术在电子显微镜下发现小鼠淋巴结生发中心早期凋亡细胞。方法用GA,PA,PLP分别固定淋巴组织,将其分别切成50μm切片,TUNEL染色,制成1μm切片光镜确认,着色部位制成超薄切片,在电镜下,进行比较观察。结果GA固定的组织中细胞核的TUNEL染色,虽然表面清晰可见,但对组织渗透性较差;PA固定的组织清晰度稍差,但渗透性最好,在电子显微镜下观察效果满意,PLP固定染色效果差,在细胞凋亡的早期,用PA染色时凋亡的细胞核内,可见尚未出现凋亡的生发中心细胞核形态学改变以及核染色质浓缩的核。结论以PA固定的组织,用包埋前技术、TUNEL染色的方法具有简便,染色清晰,易分辨,特异性强的特点,且未见标本损坏现象。     

SpermBlue®: A new universal stain for human and animal sperm which is also amenable to automated sperm morphology analysis

Abstract

Our study was aimed at exploring a simple procedure to stain differentially the acrosome, head, midpiece, and flagellum of human and animal sperm. A further prerequisite was that sperm morphology of the stained samples could be analyzed using automated sperm morphology analysis (ASMA). We developed a new staining process using SpermBlue® fixative and SpermBlue® stain, which are iso-osmotic in relation to semen. The entire fixation and staining processes requires only 25 min. Three main steps are required. First, a routine sperm smear is made by either using semen or sperm in a diluting medium. The smear is allowed to air dry at room temperature. Second, the smear is fixed for 10 min by either placing the slide with the dried smear in a staining tray containing SpermBlue® fixative or by adding 1 ml SpermBlue® fixative to the slide. Third, the fixed smear is stained for 15 min by either immersing the slide in a staining tray containing SpermBlue® stain or adding four drops of SpermBlue® stain to the fixed smear. The stained slide is dipped gently in distilled water followed by air drying and mounting in DPX® or an equivalent medium. The method is simple and suitable for field conditions. Sperm of human, three monkey species, horse, boar, bull, ram, mouse, rat, domestic chicken, fish, and invertebrate species were stained successfully using the SpermBlue® staining process. SpermBlue® stains human and animal sperm different hues or intensities of blue. It is possible to distinguish clearly the acrosome, sperm head, midpiece, principal piece of the tail, and even the short end piece. The Sperm Class Analyzer® ASMA system was used successfully to quantify sperm head and midpiece measurements automatically at either 600 × or 1000 × magnification for most of the species studied.     

Detection of S-Phase Cells in Smear Cytology Using in Vitro Bromodeoxyuridine Labeling

A technique Is described for rapid detection of S-pha?e cells of tumor tissues in smear specimens using bromodeoxyuridine (BrdU) immunostaining. Mouse NR-S1 tumors and human tumor specimens were prepared for smear cytology after incubation in RPMI 1640 culture medium containing 200 μM BrdU at 37 °C under 3 atm for 1 hr. Samples were fixed in 70% ethanol for 30 min and used immediately or air dried for 30 min. Samples were then denatured in either 4 N HC1 or 0.07 N NaOH to prepare partially single-stranded DMA. Fixation with air drying for 30 min followed by 30 min in 70% ethanol and 1 min denaturation with 0.07 N NaOH resulted in satisfactory staining quality. Cultured tumor specimens were processed for routine paraffin sections after smears were made for cytology. The labeling indices of the smear specimens and of the paraffin sections gave similar results. This technique should be useful in evaluating the cell proliferative potential of tumor tissue in smear cytology without processing paraffin sections.     

Novel zinc-based fixative for high quality DNA, RNA and protein analysis

We have developed a reliable, cost-effective and non-toxic fixative to meet the needs of contemporary molecular pathobiology research, particularly in respect of RNA and DNA integrity. The effects of 25 different fixative recipes on the fixed quality of tissues from C57BL/6 mice were investigated. Results from IHC, PCR, RT–PCR, RNA Agilent Bioanalyser and Real-Time PCR showed that a novel zinc-based fixative (Z7) containing zinc trifluoroacetate, zinc chloride and calcium acetate was significantly better than the standard zinc-based fixative (Z2) and neutral buffered formalin (NBF) for DNA, RNA and protein preservation. DNA sequences up to 2.4kb in length and RNA fragments up to 361bp in length were successfully amplified from Z7 fixed tissues, as demonstrated by PCR, RT–PCR and Real-Time PCR. Total protein analysis was achieved using 2-D gel electrophoresis. In addition, nucleic acids and proteins were very stable over a 6–14-month period. This improved, non-toxic and economical tissue fixative could be applied for routine use in pathology laboratories to permit subsequent genomic/proteomic studies.     

Comparison of DNA ploidy and nuclear size, shape and chromatin irregularity in tissue sections and smears of prostatic carcinoma

Image analysis measurements of nuclear size, shape, texture and DNA ploidy were compared in smears versus the corresponding 4-microns tissue sections, both prepared from radical prostatectomy specimens obtained from resections for prostatic cancer. Thirty-nine cases (78%) showed concordant DNA histograms between the smear and the tissue section. In six cases (12%), both preparations were nondiploid, but a tetraploid population was also present in one, but not both, of the preparations. In five cases (10%), there was a major discordance between the smear and the tissue section, with one preparation diploid and the other nondiploid. One source of discrepancy between the smear and tissue histograms was the overlapping of larger nuclei in tissue sections, which often precluded the analysis of the most atypical cells. Some tissue histograms were difficult to interpret due to wide coefficients of variation, irregular peaks and some shift from 2n in the diploid peaks. The best morphometric correlation (0.78) between the smears and the tissue sections was for the modal nuclear shape. Nuclear size and texture measurements showed poorer correlations. These findings suggest that cytologic preparations of prostatic carcinoma should be preferred for image analysis.     

PAXgene fixation enables comprehensive metabolomic and proteomic analyses of tissue specimens by MALDI MSI

An alcohol-based non-crosslinking tissue fixative, PAXgene Tissue System, has been proposed as alternative fixation method to formalin, providing superior and morphological preservation. To date, metabolites have not been assessed in PAXgene-fixed tissues. The study focuses on a comparison between PAXgene and standard formalin fixation for metabolomic analysis by MALDI mass spectrometry imaging. Therefore, fifty-six samples from seven mice organs were fixed with PAXgene (PFPE) or formalin (FFPE), embedded in paraffin, and processed to a tissue microarray. PAXgene was able to spatially preserve metabolites in organs achieving an overlap of common metabolites ranging from 34 to 78% with FFPE. Highly similar signal intensities and visualization of molecules demonstrated negligible differences for metabolite imaging on PFPE compared to FFPE tissues. In addition, we performed proteomic analysis of intact proteins and peptides derived from enzymatic digestion. An overlap of 33 to 58% was found between FFPE and PFPE tissue samples in peptide analysis with a higher number of PFPE-specific peaks. Analysis of intact proteins achieved an overlap in the range of 0 to 28% owing to the poor detectability of cross-linked proteins in formalin-fixed tissues. Furthermore, metabolite and peptide profiles obtained from PFPE tissues were able to correctly classify organs independent of the fixation method, whereas a distinction of organs by protein profiles was only achieved by PAXgene fixation. Finally, we applied MALDI MSI to human biopsies by sequentially analyzing metabolites and peptides within the same tissue section. Concerning prospective studies, PAXgene can be used as an alternative fixative for multi-omic tissue analysis.     

Suppression of connective tissue impregnation in a silver technique for demonstrating nerve fibers.

A tissue pretreatment is introduced which effectively suppresses the silver impregnation of connective tissue and nonspecific background elements in peripheral nerve. The result is a selective impregnation of nerve fibers. The procedure utilizes fresh frozen sections and can be used with the Holmes (1947) or Bodian (1936) techniques. Fresh frozen sections are cut at 10 microns, mounted on slides and air dried for 5 minutes. They are fixed for 30 minutes in formol-sublimate (10% formalin saturated with mercuric chloride) and then placed into 0.5% iodine in 70% alcohol for 5 minutes followed by bleaching in 2.5% sodium thiosulfate for 2 minutes. After washing in running tap water for 10 minutes and a brief rinse in distilled water, impregnation is accomplished by the Holmes (1947) or Bodian (1936) procedure beginning with the step containing the aqueous silver solution. The results show an absence of impregnation of connective tissue and nonspecific background. The technique is simple, rapid, and, by utilizing fresh frozen sections, can be used for other histological and histochemical purposes. Several experiments were done to determine the causes of the connective tissue and background suppression. The air drying step was omitted; the sections were fixed in formalin without mercuric chloride; and the formol-sublimate fixation time was increased. The results suggest that connective tissue impregnation is suppressed by the use of mercuric chloride in the fixative and that the background suppression is related to the short fixation time with formolsublimate.     

Computer assisted morphometric analysis of ram sperm heads: evaluation of different fixative techniques

The recent development of automated systems for morphometric sperm head analysis has provided a series of objective parameters which have facilitated the standardization of morphological semen evaluation. This current work attempts to establish the optimum fixing conditions for the morphometric characterization of ram spermatozoa. Ejaculates were obtained from 5 Merino rams used for periodic collection of semen and were diluted at 1:50 with TEST medium. Air-dried smears were fixed either in ethanol-ether (1:1), 50% methanol, 2% glutaraldehyde or SUZA fixative, in which case the smear was pretreated with chloramine. The samples were then stained with commercial kit Hemacolor. Once the preparations had been mounted, they were analyzed with the Sperm Class Analyzer automatic sperm morphometry analysis system (ASMA). The minimum number of sperm cells analyzed per sample was 100. The parameters evaluated were the area, perimeter, length, width, shape factor and mass. The results showed significant differences in sperm head dimensions between the 4 fixation techniques, with the lowest values for all parameters corresponding to the SUZA fixative, followed by glutaraldehyde, methanol, and finally ethanol-ether. In addition, there were significant variations between animals. It can, therefore, be concluded that the working protocol must be defined when performing morphometric analysis of ram semen and that the results obtained under different conditions of fixation cannot be entirely extrapolated. Equally, the high variability among individuals suggests that, in a species like the ram with a low index of teratozoospermia, there is a need for a revision of the classic definition of normality, which should include morphometric data.     

Comparison of fixatives for maximal retention of radiolabeled glycoconjugates for autoradiography, including use of sodium sulfate to release unincorporated [35S]-sulfate

Previous studies have used [35S]-sulfate as a specific marker to autoradiographically localize sulfated glycosaminoglycans, proteoglycans, and glycoproteins. Embryonic chicks were labeled with [35S]-sulfate, followed by previously reported routine fixation and processing techniques. Subsequent processing revealed loss of radiolabeled macromolecules and retention of unincorporated label in the tissue, using these procedures. Biochemical analysis after various fixation and processing procedures demonstrated that an additional agent, such as cetylpyridinium chloride, was necessary in the fixative to retain the highly aqueous soluble sulfated macromolecular components. Molecular sieve chromatography was used to monitor digestate solutions for the identity of glycosaminoglycans and proteoglycans as indicated by selective enzymatic removal. Retained unincorporated [35S]-sulfate could be completely removed by rinsing the tissue in dehydration solutions containing exogenous sodium sulfate. This new procedure ensures the quantitative retention of sulfate labeled macromolecules in fixed tissue with the complete removal of unincorporated radiotracer, both of which are necessary for meaningful autoradiography.     

A fixative for use in muscle histochemistry

A fixative solution that preserves the activity of some relevant enzymes in muscle histochemistry is described. Portions of human muscle biopsy specimens and selected murine muscles were fresh frozen or placed in the fixative at room temperature for up to 1 month before freezing. Cryostat sections of fresh frozen and fixed frozen tissue were assayed for nicotinamide adenine dinucleotide phosphate (NADH)-tetrazolium reductase (NADH), several adenosine triphosphatases (ATPases), myoadenylate deaminase (MD), and phosphorylase. NADH, ATPase, and MD activity were preserved following fixation but phosphorylase was not preserved. Murine spleen and kidney were similarly tested for acid phosphatase (acid phos), alkaline phosphatase (alk phos), and nonspecific esterase (NSE). Alk phos activity was preserved but acid phos and NSE activity were significantly reduced following fixation. This fixative is useful in some circumstances for processing or shipping human muscle biopsy specimens and experimental tissues.     

Alteration of immunoglobulin-bearing lymphoma cells by fixation.

Four large cell lymphomas known to be monoclonal B-cell proliferations were studied with immunofluorescent and immunohistochemical methods for the detection of kappa- and lambda-light chains. Frozen sections of lymphoma tissues as well as formalin and B-5-fixed tissues embedded in paraffin were studied. Both immunofluorescent and immunohistochemical methods gave similar results on frozen sections; however, a number of discrepancies were noted between the results obtained on fixed tissues and those obtained on frozen tissues. In an effort to identify a fixative which did not alter immunoglobulin (Ig), mouse lymph nodes were fixed in different fixatives before Ig detection; but all of the fixatives tested destroyed the Ig present on normal cortical B lymphocytes. Immunoglobulin-bearing normal and neoplastic lymphocytes are better detected on frozen sections than on paraffin sections after routine fixation.     

Morphometry, densitometry and pattern analysis of plastic-embedded histologic material from urothelial cell carcinoma of the bladder.

An image analysis method of grading histologic sections of bladder carcinoma was tested. The method was new in four respects. First, for fixation of the biopsies a coagulant fixative was used. Second, 2-microns plastic sections were used to ensure the reproducibility of nuclear imaging. Third, a new stereologic approach was used for calculation of the nuclear volume and DNA content. Fourth, for the classification rule the morphometric, densitometric and texture features were used in concert. The IBAS 2000 instrument was used for the measurements. Texture analysis of the chromatin patterns was performed using Markovian texture features. Using discriminant analysis, of 22 parameters, 2 morphometric, 2 densitometric and 3 texture features were selected for the classification rule. With them, 89% of the bladder carcinomas were correctly classified into the three grades. All grade III tumors were classified correctly. Among the features tested, the densitometry of the DNA had the highest F values. All of the grade III tumors and 45% of the grade II tumor group had DNA histograms indicating aneuploidy. This study showed that plastic-embedded material is well suited to morphometry and densitometry and can be used for quantitative grading of bladder carcinoma.     

Electron dense artefactual deposits in tissue sections: the role of ethanol, uranyl acetate and phosphate buffer.

The occurrence of electron dense deposits in sections of aldehyde-fixed tissue prepared for transmission electron microscopy has been attributed to a number of conflicting factors. In an attempt to clarify this, the precipitating effect of different combinations of phosphate or cacodylate buffer, glutaraldehyde, ethanol and uranyl acetate was investigated in test tubes. As a preliminary investigation the combination of phosphate buffer, ethanol and uranyl acetate was investigated in heart and kidney tissue fixed in glutaraldehyde with or without postosmication. The essential factors in the formation of electron dense deposits in these tissues appear to be phosphate buffer, ethanol, and uranyl acetate, although glutaraldehyde may contribute in some way. The nature and intensity of the deposits seem to vary with the sequence of combination of these factors. Osmium did not appear to be an essential factor in the reaction since deposits were observed in both osmicated and unosmicated tissue. To avoid such deposits, a postosmication distilled water wash for 20 to 30 min followed by en bloc staining with aqueous uranyl acetate is advised if phosphate buffer is used as a fixative vehicle or buffer wash after the primary fixative.     

The influence of protease digestion and duration of fixation on the immunostaining of keratins. A comparison of formalin and ethanol fixation

The effect of three proteases--trypsin, pepsin, and pronase--on the immunohistochemical staining of keratins with a broad-spectrum monoclonal antibody was investigated in paraffin sections of formalin and ethanol-fixed tissues by means of the peroxidase-antiperoxidase method. Both the length of exposure to the fixative and the duration of proteolysis were varied over a wide range. Ethanol-fixed tissues showed excellent preservation of the antigenicity of keratins, and no appreciable differences in immunostaining related to the length of fixation were found. The use of proteolytic enzymes did not improve these results; on the contrary, it caused rapid tissue disintegration. Formalin-fixed epithelial tissues stained weakly or failed to stain unless they were treated with a proteolytic enzyme. The optimal length of proteolysis varied with the degree of fixation; tissues that were fixed for long periods of time in formalin required longer exposure to a proteolytic enzyme and were more resistant to digestion than were tissues that were fixed briefly. No significant advantage of one protease over another was found in this study. We conclude that a proteolytic step must precede immunostaining for keratins if the tissue is fixed in formalin, but that the digestion period must be adjusted according to the length of exposure to the fixative. The superiority of alcohol over formalin fixation for the preservation of the antigenicity of keratins is confirmed by this study.