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PubMed1996

A comparative study of the transition between predentin and dentin, using various preparative procedures in the rat.

The appearance of the junction between predentin and dentin of rat incisors was investigated after chemical fixation (aldehyde), physical fixation (high pressure freezing and freeze substitution) and by histochemistry. Physical fixation revealed a 1-2 micron wide intermediary zone, in which only the collagen fibers were mineralized. In dentin, which looked denser and more homogeneous, both collagen fibers and intercollagenous spaces were mineralized. The intermediary layer could not been seen after aldehyde fixation. When cationic dyes were used during fixation in order to retain proteoglycans, a 0.5-5 micron border zone located at the junction between predentin and dentin was densely stained on the dentin side, whereas in dentin only the interglobular network displayed electron density. The periodic acid-thiocarbohydrazide-silver proteinate reaction, visualizing glycoproteins, and the phosphotungstic acid/chromic acid mixture, which reveals glycoproteins and phosphorylated proteins, produced extensive staining of the transitional zone located at the dentin edge, whereas staining was weaker in dentin. These morphological and histochemical investigations support the existence of an intermediary zone in which mineralization occurs. This transitional zone between predentin and dentin has specific properties; therefore we propose it be termed metadentin.

Aldehydes↗
PubMed1992

[Nucleolus organizer regions in pathomorphologic tumor diagnosis].

Morphology and biological behaviour of tumours are closely related to each other and are the main objects of diagnostic tumour pathology. Anaplasia, for example, a well-known cytomorphological criterion of malignancy, is however only an indirect sign of impaired cellular growth control mechanisms. Nowadays, it is a goal of cancer research to investigate these regulatory processes in order to elucidate new diagnostic criteria of malignancy. Silver staining of nucleolar organizer regions (AgNORs) basically meets this new concept. NORs (rDNA) encode for ribosomal RNA and are thus of central importance for the protein synthesis of a cell. Silver stains NOR associated proteins and indicates activated rDNA. Therefore, AgNOR staining covers the proliferative potential of a given cell or a tumour respectively. A prerequisite of reliable application of the AgNOR method to diagnostic tumour pathology is standardization of the technique, which should be based upon scientific results obtained by basic research. Accordingly, in interphasic cells AgNORs are primarily substructures of nucleoli corresponding to the fibrillar centers at the ultrastructural level. This has been shown to be an important criterion to determine the quality of the silver staining reaction. Most artifacts result from inadequate tissue preservation (fixation), unspecific argyrophilic structures and the staining procedure itself. They should either be excluded or--when ever possible--controlled by a proper staining technique, i.e. adjustment to the stainability of every individual specimen. In this respect, digital image analysis proved to be a useful tool. The mean size of AgNORs in small lymphocytes of the same tissue sample is an equivalent of the argyrophilia of a given histological and cytological specimen (internal staining standard). By use of such a standardized staining technique a high diagnostic accuracy is achieved. The proliferative potential of a cell or a tumour can be determined by number, size, staining intensity and distribution of the silver stained dots. In histological tissue sections the quotient of the mean number of AgNORs/cell and the mean area of one AgNOR dot/cell has been shown to be a very sensitive diagnostic parameter. In cytological specimens determination of the total silver stained area per cell is superior to counting of AgNORs simply by eye. Normal and malignant tissue can reliably be discriminated by these morphometric values. In addition, the malignancy potential of the different preneoplastic lesions, e.g. of the urinary bladder, can precisely be described. Flat urothelial lesions with moderate cytological atypia (D2) exhibit AgNOR values in the range of well differentiated papillary carcinomas.(ABSTRACT TRUNCATED AT 400 WORDS)

Biomarkers, Tumor↗
PubMed1994

Quantification of cell loss during bronchoalveolar lavage fluid processing. Effects of fixation and staining methods.

Discrepancies have been reported in differential cell counts according to the diverse processing methods used in bronchoalveolar lavage (BAL) fluid management. The differences have proved to be mainly the result of selective lymphocyte loss, while the exact mechanisms of the phenomenon remain controversial. Observing a similar variation in differentials from differently stained identical smears, we quantified the cell loss due to staining procedures from 45 consecutive satisfactory BAL procedures. To do this, we compared relative lymphocyte recovery on neat pooled lavage in a hemocytometer with that from smears and cytopreps fixed and stained in different ways. We found (1) A significant lymphocyte loss (p < 0.05) whatever the staining method. (2) Different methods of fixation and staining lead to considerable variation in differentials from slides otherwise identically managed. The loss is higher during air-drying fixation followed by staining with an aqueous medium such as Diff-Quik than on spray-fixed slides stained in an alcohol medium such as Papanicolaou stain. (3) The effect of lymphocyte loss on differentials is more important when the initial lymphocytosis is less than 35%, and decreases to nonsignificance when it exceeds 70%. The role of cytocentrifugation or other manipulations in cell loss probably has been overestimated because unknown effects of staining methods were also attributed to these manipulations. We suggest that lymphocyte loss could arise from poor adherence on slides, which is exacerbated during aqueous staining if no artifice (e.g., spray fixation), is used to hold them. Thus, the definition of the long-awaited standard procedure for an accurate differential count of BAL fluid must take into account fixation and staining methods.

Bronchoalveolar Lavage Fluid↗
PubMed1994

An investigation into the use of protein cross-linking agents as cell fixatives for confocal microscopy.

A variety of compounds which are known to cross-link proteins have been tested as possible cell fixatives for confocal microscopy. The criteria for good fixation that were used for this work were that the fixative should make the cells resistant to lysis and the loss of protein from the cell under hypotonic conditions. In addition, fixation should not change the shape nor the volume of the cell. Of the compounds tested, one compound, EDC, appeared to have an excellent potential as a cell fixative and has the additional advantage that it does not require amino groups for its crosslinking activity. Another compound, SPDP, appeared to have some potential as a readily reversible fixative by virtue of its disulfide bridge.

Cell Size↗
PubMed2000

Mucosal biopsy techniques and interaction with the pathologist.

The endoscopy era made it possible to see many of the diseases that were being treated by clinicians. The use of endoscopic biopsy further enhanced that ability. This article illustrates how gastrointestinal biopsy and other practices can be improved so that patients benefit more than they might otherwise. This article focuses on pinch biopsy forceps technique and on dialogue with the pathologist.

Biopsy↗
PubMed1996

Hepatocellular carcinoma in patients from northern Sweden with acute intermittent porphyria: morphology and mutations.

More than a decade ago an association between acute intermittent porphyria (AIP) and hepatocellular carcinoma (HCC) was reported, but still the cause of the increased prevalence is unknown. Paraffin sections of formalin-fixed HCC from 17 AIP patients were reexamined and also screened for relevant mutations using several methods. The tumor diagnosis was verified, and in several cases precirrhosis and cirrhosis were also found. The clinically founded AIP diagnosis was verified at the gene level in most cases, demonstrating the Norrland type of mutation, i.e., G(593)-to-A substitution in codon 198 of the porphobilinogen deaminase (PBGD) gene. The second allele was neither mutated nor missing, contradicting the possibility that the PBGD gene might function as a tumor suppressor gene. Subsequent sequencing showed that cases not cleaved by the restriction enzyme NheI lacked the specific Norrland mutation. In recent years, selective mutations at codons 249 and 166 of the p53 gene have been described in HCC associated with aflatoxin and hepatitis B virus. In our area, with low exposure to those agents, no mutations in codon 249 were found, and mutation in codon 166 was excluded in all tumors except one; no traces of hepatitis B DNA were observed. Nor did we find mutations in H-ras 12 or 61. Intrinsic aberrations in AIP, including reduced heme synthesis and endogenous oxidative damage to DNA, may incite carcinogenic mutations elsewhere in the genome of liver cells. The increased cell proliferation coupled to precirrhosis and cirrhosis perhaps represents promotion in the initiation-promotion sequence of hepatocarcinogenesis.

Adenine↗
PubMed1996

Influence of tissue preparation on the high-frequency acoustic properties of normal kidney tissue.

The influence of various tissue preparations on the acoustic properties of normal kidney tissue at high frequencies was investigated. Eight surgically excised normal kidney tissue specimens were classified into three groups: (i) fresh, frozen section, (ii) formalin-fixed, frozen section and (iii) formalin-fixed, paraffin section. Scanning acoustic microscopy operating in the frequency range of 100-200 MHz was used to display the two-dimensional distribution of attenuation constant and sound speed. Our results indicate that (i) there is no significant variation in both acoustic parameters between the three tissue groups, (ii) fixation by 10% formalin produces no significant change in the acoustic parameters, (iii) in fat-free tissue regions, the acoustic parameters are independent of preparation method and (iv) frozen sections must be used to assess the acoustic parameters in fat-rich tissues.

Histocytological Preparation Techniques↗
PubMed1993

Fixation with formaldehyde induces expression of activation dependent platelet membrane glycoproteins, P selectin (CD62) and GP53 (CD63).

Platelet activation in vivo is important in the pathogenesis of thrombosis. Accurate measurement is difficult due to artefactual in vitro preparation related activation. This can be overcome by using whole blood techniques, such as flow cytometry. However, there is little consensus on methods of platelet preparation, procedures for use of fixation, or the optimal types/amounts of fixative. The use of unfixed platelets has received little attention. We describe a series of experiments comparing platelet activation antigen expression detected by flow cytometry in fixed and unfixed samples. Formaldehyde increased the expression of both CD62 and CD63. We recommend that fixation with formaldehyde should not be used when studying platelet activation.

Antigens, CD↗
PubMed1996

Improved ultrastructural preservation of yeast cells for scanning electron microscopy.

The processing of yeast cells for scanning electron microscopy by conventional sequential fixation with glutaraldehyde and osmium tetroxide and subsequent dehydration and critical point-drying caused pronounced deformation and visible shrinkage in all basidiomycetous and ascomycetous yeast strains studied. The mean cell diameter decreased to nearly 60 and 70%, respectively. After an additional sequential fixation with 1% tannic acid and 0.5% uranyl acetate the cell shrinkage was significantly reduced, but the most important result was a considerable reduction of wrinkling and deformation of the yeast cells.

Microscopy, Electron, Scanning↗
PubMedDate not supplied

Rehydration of air-dried smears. An alternative method for cytologic analysis of exfoliative cells.

OBJECTIVE: To compare the cytologic characteristics of gastric mucosal cell smears prepared by air drying and rehydration prior to alcohol fixing with cells wet fixed in alcohol. STUDY DESIGN: Gastric mucosal cells were obtained from 55 consecutive patients undergoing gastroscopy. Paired smears were made, one immediately fixed in 95% ethanol for 20 minutes (wet fixed [WF]) and the other air dried for at least 20 minutes prior to rehydration with normal saline for 30 seconds and fixation in 95% ethanol for 20 minutes (air dried/rehydrated/fixed [ARF]). Both slides were stained by the Papanicolaou method. Coded slides were examined blind and graded 1 (superior), 2 (satisfactory) or 3 (poor) with respect to staining of chromatin, nuclear membrane, nucleoli, cytoplasm/cell border and group morphology. Histology confirmed a benign disease process or normal mucosa. RESULTS: Comparing grade 1 versus grades 2 and 3, ARF slides were significantly better than WF slides for all cytologic features (P < .05). Comparing grade 1 and 2 versus grade 3, there was no significant difference between ARF and WF slides (P > .05) (chi 2 analysis). CONCLUSION: The cytologic features of ARF smears of gastric cells were equal or superior to those of WF smears. This method of preparing smears is simpler and avoids some of the problems of ethanol fixation of wet smears.

Attitude of Health Personnel↗
PubMed1997

Structural comparisons of cell organelles and nuclei in rat hepatocytes with three different fixation techniques.

The morphological properties of cell organelles and nuclei in rat hepatocytes were compared, when the hepatocytes were either chemically fixed in glutaraldehyde and osmium (GA-Os), chemically fixed only in osmium (Os-2h) or physically fixed with a microwave-irradiated osmium (Mw-Os) fixative for a short duration. Judging from the fine structure, the mitochondria and rER were best preserved after physical fixation; because of the smooth surfaces and regular open contours, but both organelles shrank in the two chemical fixatives. The Golgi complexes and peroxisomes of the hepatocytes were not markedly changed by any of the fixative procedures. However the lysosomes and sER were changed to some extent after physical fixation. In the nucleus, the two kinds of chromatins and the nucleolus were best preserved with physical fixation. In conclusion, physical fixation is a very simple and fast procedure that is convenient for routine electron microscopy if some attention is paid to the morphological changes.

Animals↗
PubMed2000

Microwave processing for scanning electron microscopy.

The normal processing of biological samples for Scanning Electron Microscopy, includes treatment with aldehyde (1 to 2 hours), postfixation with Osmium (1 hour), followed by dehydration in a ascending grade of ethanol (30 a 100%), 10 to 15 minutes in each step, and finally drying. This procedure takes at least 8 hours. In this work, samples of mosquitoes (Aedes), protozoa (Tritrichomonas muris), bacteria (Clostridium oceanicum), murine liver, and small intestine were processed in the same manner in a domestic microwave oven for two minutes at 20% of its maximum power. The complete procedure from the initial fixation to dehydration in 100% ethanol was reduced to one hour with good preservation of the ultrastructural details of the specimens.

Aedes↗
PubMed1998

Preferential silver reaction of nucleolar regions adjacent to fibrillar centers in ring shaped nucleoli of leukemic lymphocytes.

Silver stained proteins (SSPs) characteristic for interphasic nucleolus organizer regions (NORs) associated with fibrillar centers (FCs) and adjacent nucleolar regions of ring shaped nucleoli in leukemic lymphocytes exhibit a different sensitivity to the mild acid extraction including that with HCl. Such extractions permit a preferential visualization of fibrillar centers adjacent regions (FCARs) which are believed to represent sites of the ribosomal RNA (rRNA) transcription. The resistance of SSPs in FCARs to the extraction with HCl seems to be due to their binding to other components present in these regions. The extractibility of SSPs with HCl was influenced by the fixatives used. The largest resistance of SSPs to the extraction with HCl was noted after fixation with glutaraldehyde. In contrast, the largest extractibility of these proteins was observed after fixation with unbuffered formaldehyde.

B-Lymphocytes↗
PubMed2002

Inadequate formalin fixation decreases reliability of p27 immunohistochemical staining: probing optimal fixation time using high-density tissue microarrays.

Immunohistochemical analysis of molecular targets in clinical tissues is increasingly becoming central to our ability to render diagnoses, to predict prognosis, to select patients for appropriate therapies, and to provide surrogate end points for therapeutic monitoring. For example, reduction of immunohistochemical staining for the cyclin-dependent kinase inhibitor p27(Kip1) has been proposed as a potential prognostic biomarker in prostate, breast, and gastrointestinal tumors. We observed that with our standard formalin fixation in rapidly processed (same-day) radical prostatectomy specimens, there is often a gradient of p27(Kip1) staining in normal prostate epithelium, with more staining near the periphery and less staining toward the center of the sample. This raised the hypothesis that the reliability of staining for p27(Kip1) is decreased in inadequately fixed tissues. The implications of this, if true, are that many studies using p27(Kip1) for prognostic purposes may be subject to unpredictable artifacts, and hence unreliable results, if the fixation of the specimens is not well controlled. The objectives of the present study were (1) to formally test the hypothesis that inadequate fixation time is responsible for apparent loss of p27(Kip1) nuclear staining and (2) to test a recently proposed method for improving the uniformity of immmunohistochemical staining using formalin injection. Prostate tissue sections from radical prostatectomy specimens were either processed immediately (zero time fixation) or fixed for 1, 2, 3, or 8 days in 10% neutral buffered formalin before processing into paraffin. To assure identical antigen retrieval and immunohistochemical staining conditions for specimens fixed for different lengths of time, 2 high-density tissue microarrays (TMAs), containing 564 tissue samples (0.6 mm in diameter) were constructed. Based on an estimate of the percentage of nuclei in normal prostatic epithelial secretory cells with strong staining, quality of p27(Kip1) staining was graded in a blinded fashion with respect to fixation time. There was a significant increase in the percentage of cores that were scored as "strong" as fixation time increased from 0 (same-day processing) to 1 or more days (P <.0001). Interestingly, even at 8 days of fixation, there was excellent staining that was superior to the same-day processing. Based on these results, we conclude the following: (1) for large clinical specimens that have been fixed briefly to decrease diagnostic turn-around time, the reliability of interpretation of immunohistochemical staining may be quite limited; (2) for p27(Kip1), decreased antigen staining as a result of the widely held concept of "overfixation" is much less of a problem than "underfixation"; (3) formalin injection produces a marked improvement in staining for several markers, including p27(Kip1); and (4) high-density TMAs, which assure identical test conditions, provide an excellent platform on which to evaluate the effects of tissue fixation on immunohistochemical staining.

Cell Cycle Proteins↗
PubMed1983

[Proton relaxation characteristics of human and animal tissue in vitro. Changes during autolysis and fixation].

The results of measurements of proton relaxation times of various tissues from rats, pigs and humans are reported; these were obtained by a resonance spectroscope at 20 MHz and 40 degrees C. There were specific differences in both relaxation times (T1 and T2) of the liver and spleen. There was a difference of more than 150 ms in the longitudinal relaxation time between grey and white cerebral tissue. Autolytic changes show an increase in both relaxation times. Fixation produced a reduction in T1 only. The significance of these findings for NMR tomography is discussed.

Animals↗
PubMed1995

Application of a multiple fixation regimen to study the adaptive response to ionizing radiation in lymphocytes of two human donors.

The majority of experiments studying the adaptive response using chromosomal aberrations have been performed with proliferating lymphocytes. It is known that lymphocytes have variable cell cycle transit times and it has been pointed out that in such cases aberration scores obtained from a single harvest are not very meaningful because cells harvested together in metaphase at any one time after irradiation were in different parts of the cell cycle at the time of irradiation. The scored sample will thus always contain a mixture of cells having different radiosensitivities and any variations of cell proliferation will influence the aberration score. In order to get a more representative aberration score a multiple fixation regimen was applied to lymphocytes of two human donors. Cells receiving the adapting + challenging and the challenging dose were fixed at three intervals after the challenge. In lymphocytes of donor 1 no adaptive response was seen at any fixation time in two experiments. In lymphocytes of donor 2, however, a reduction of aberration frequencies was seen, but at different fixation times in the two experiments. In a third experiment, no adaptive response was detected. It is concluded that the response observed at some fixation times in lymphocytes of donor 2 is rather a result of some phenomenon associated with variations of cell cycle kinetics than of induced radiation resistance.

Adaptation, Physiological↗