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PubMed1975

Biochemical aspects of aldehyde fixation and a new formaldehyde fixative.

In this paper, it is assumed that tissue fixation is a process in which the proteins become less soluble and catabolic reactions stop. With this definition in mind, 2.5 and 5% glutaraldehhde and 4% formaldehyde in 0.1 M potassium phosphate buffer, pH 7.4, were compared with a new fixative, bicarbonate-formaldehyde. The following results were obtained. (I) With 2.5 and 5% glutaraldehyde, the solubility of tissue proteins were not decreased unifromly, and tissue glycogen was poorly preserved. (2) 4% formaldehyde in potassium phosphate buffer gave relatively good results. (3) Bicarbonate-formaldehyde decreased the solubility of tissue proteins reliably and preserved tissue glycogen perfectly. Histologically, it yielded excellent results. Since glutaraldehyde alters the properties of proteins substantially (Hopwood, 1972; Habeeb & Hiramoto, 1968), and since the natural appearance of tissues depends on native tissue proteins, formaldehyde-containing fixatives, in particular bicarbonate-formaldehyde, are preferable to glutaraldehyde-containing fixatives for all tissue preparative techniques. However, it is important that the fixation time in formaldehyde is kept short.

Aldehydes↗
PubMed1979

Mitotic activity and delay in fixation of tumour tissue. The influence of delay in fixation on mitotic activity of a human osteogenic sarcoma grown in athymic nude mice.

The purpose of the present investigation was to study the effect of delay in fixation on the mitotic activity in tumour tissue. A human osteogenic sarcoma, especially suitable for counting of mitoses, grown in athymic nude mice, was fixed with varying delay and the mitotic, prophase, metaphase and ana-telophase indices were determined. An almost exponential decline of the mitotic index was observed with a reduction to 49.4% and 15.0% after respectively 60 and 180 minutes. The proportional incidence of prophases, metaphases and ana-telophases changed so that a relative accummulation of advanced phases occured during the 180 minutes of observation. It is concluded that delay in fixation of a magnitude, which is not uncommon in routine surgical pathology, may allow the majority of mitoses to terminate, resulting in unreliable assessments of mitotic activity.

Adult↗
PubMed2006

The use of rapid assessment of enteric ICC and neuronal morphology may improve patient management in pediatric surgery: a new clinical pathological protocol.

Alterations in interstitial cells of Cajal (ICC) distribution and density may seriously influence gut motility. We and others have documented a disturbance of ICC and neurons in Hirschsprung's disease (HD), intestinal ischemia, and inflammation. The ability to remove intestine with permanent dysmotility improves significantly the prognosis. This may be important in the developing intestine especially in HD. More complete pathological information increases the accuracy of surgical decisions. Therefore we sought to develop a rapid, intraoperative immunohistochemical protocol for ICC and neurons in surgical specimens for routine diagnostic and therapeutic assessment of pediatric patients. To date, cKit is the only reliable immunohistochemistry for ICC identification. There are multiple antibodies in use for neuronal identification. A comparison of fixation methods and immunostaining using cKit and multiple intestinal neuronal antibodies was done. Fresh segments of surgically resected intestine were sectioned and stained using antibodies for ICC cell identification (anti-cKit) and for neuronal characterization. By carefully changing tissue fixation methods, different neuronal antibodies were tested to determine an optimal rapid protocol. Each suggested protocol was tested on normal and pathological intestinal tissue and compared to the previous overnight immunostaining of the same tissue. A new rapid tissue fixation and immunostaining protocol using cKit for ICC identification and NF 68 was developed. By employing this protocol, we could obtain ICC and neuro-immunohistochemistry in unfixed frozen sections within 1 h with tissue vibration to diminish the time for immunostaining. Without vibration the protocol takes 3 h. ICC and enteric neuronal changes could be readily observed and the quality of staining was comparable to standard immunohistochemistry. Each gut pathology displayed characteristic changes in ICC and neuronal density/distribution in the affected bowel. The time-scale of the 1-h immunoprocessing is still longer than the standard clinical pathological "quick" sections using H&E staining; however, the protocol duration is within the surgery timescale. Standard H&E stain used in combination with our rapid neuronal and ICC immunohistochemistry protocol enables a fast, comprehensive, and accurate assessment of the pathophysiology of signaling networks controlling gut motility while the patient is still in the operating room. We propose that the addition of this simple and rapid immunohistochemical assessment in the pathologist evaluation of surgical specimens would result in a more complete characterization for diagnosis and prognosis of the pediatric patient. Specifically, we propose that this test will differentiate good versus poor prognosis HD patients based on their neuron/ICC ratio and present a rapid, standardized method for use in general pathology.

Colon↗
PubMed2006

A citric acid-based hydroxyapatite composite for orthopedic implants.

We describe a novel approach to process bioceramic microparticles and poly(diol citrates) into bioceramic-elastomer composites for potential use in orthopedic surgery. The composite consists of the biodegradable elastomer poly(1,8-octanediol-citrate) (POC) and the bioceramic hydroxyapatite (HA). The objective of this work was to characterize POC-HA composites and assess the feasibility of fabricating tissue fixation devices using machining and molding techniques. The mechanical properties of POC-HA composites with HA (40, 50, 60, 65wt.%) were within the range of values reported for tissue fixation devices (for POC-HA 65wt.%, S(b)=41.4+/-3.1, E(b)=501.7+/-40.3, S(c)=74.6+/-9.0, E(c)=448.8+/-27.0, S(t)=9.7+/-2.3, E(t)=334.8+/-73.5, S(s)=27.7+/-2.4, T(s)=27.3+/-4.9, all values in MPa). At 20 weeks, the weight loss of POC-HA composites ranged between 8 and 12wt.%, with 65wt.% HA composites degrading the slowest. Exposure of POC-HA to simulated body fluid resulted in extensive mineralization in the form of calcium phosphate with Ca/P of 1.5-1.7 similar to bone. POC-HA supported osteoblast adhesion in vitro and histology results from POC-HA samples that were implanted in rabbit knees for 6 weeks suggest that the composite is biocompatible. Synthesis of POC-HA is easy and inexpensive, does not involve harsh solvents or initiators, and the mechanical properties of POC-HA with 65wt.% HA are suitable for the fabrication of potentially osteoconductive bone screws.

Animals↗
PubMed1977

Combination operant conditioning--liquid nitrogen immersion chamber for studying neurotransmitter systems and behavior.

Studies of neurochemical events associated with behavior require a method of tissue fixation that is rapid and does not itself produce neurochemical changes. An apparatus is described that permits immediate immersion of an unrestrained behaving animal into liquid nitrogen. This method of tissue fixation has the greatest versatility for studying multiple neurotransmitter systems. In addition to the measurement of neurotransmitter content and turnover, investigation of neurotransmitter receptors, enriched nerve ending fractions and enzyme activities are possible. The operant conditioning-liquid nitrogen immersion chamber described here can be used for studying these neurotransmitter systems as they relate to animal's responding on operant schedules of reinforcement.

Animals↗
PubMed2002

Laser microdissection and gene expression analysis on formaldehyde-fixed archival tissue.

BACKGROUND: Analysis of renal biopsies is currently based on histological recognition of typical structural patterns and immunohistological detection of protein expression alterations. Both can be performed using formaldehyde as the tissue fixative. As a consequence of recent advances in molecular medicine, mRNA expression analysis may offer an attractive option to obtain functionally relevant information. However, quantification of mRNA expression in human renal biopsies thus far has not been possible in formaldehyde-fixed tissue. METHODS: The present study evaluated a recently reported mRNA extraction protocol. Using this approach gene expression analysis could be performed on formaldehyde-fixed archival renal tissues by laser microbeam microdissection, laser pressure catapulting and real time reverse transcription-polymerase chain reaction. RESULTS: For an initial feasibility study, the expression of two chemokines (IP-10 and RANTES) in renal transplant rejection was examined. Induction of protein expression in allografts undergoing rejection was demonstrated for both chemokines by immunohistochemistry. The mRNA expression alterations in the defined renal compartments of glomeruli, vessels and tubulointerstitium were quantified using laser microdissection from formaldehyde-fixed, paraffin-embedded or frozen tissue sections. A pronounced increase of mRNA expression compared to controls was demonstrated for IP-10 as well as RANTES with both tissue-processing protocols. CONCLUSIONS: Using formaldehyde as the tissue fixative, information on the disease process can now be obtained by histological, immunohistochemical and gene expression techniques. In the future this may allow the study of activated molecular programs in routine renal biopsies as well as archival tissue samples.

Adolescent↗
PubMed1994

Quantitative immunohistochemical assay for hormonal receptors: technical aspects and biological significance.

Over 250 breast carcinomas were studied in order to establish whether or not quantitative immunohistochemical assays (ICA) for estrogen and progesterone receptors (ER and PR) with computer-assisted image analysis could favorably compare with standard cytosolic assays. Initially, variable antigenic preservation secondary to improper tissue fixation and processing led to irregular receptor preservation and unevenly stained areas indistinguishable from true intratumor antigenic heterogeneity. As a direct consequence of the field selections chosen for analysis, assay reproducibility was less than optimal. Proper tissue fixation and handling eliminated most of the irregular staining; selection of fields to analyze became less cumbersome and more reproducible. Differences in staining intensity due to minimal variations in the ICA also resulted in difficult reproducibility. Standardizing the technique and using an automatic stainer notably eliminated that problem. The second and equally important question was to establish if quantitative ER-ICA had relevance as a predictor for prognosis. The Kaplan-Meier product limit estimator for quantitated ER values and Cox regression for risk of mortality and disease progression were performed. The results obtained discriminated high- and low-risk groups for overall survival (p = 0.016) better than the dextran-coated charcoal assay. Elimination of two major obstacles and proof of the predictive value of quantitative ICA has transformed the assay into a valid alternative to cytosolic methods; however, before that takes place it is critical to establish standard procedures for both ICA and quantitation so interlaboratory variability is reduced to a minimum.

Adult↗
PubMed1991

Neurofilament protein triplet immunoreactivity in the dorsal root ganglia of the guinea-pig.

Immunoreactivity for the neurofilament protein triplet was investigated in neurons of the dorsal root ganglia of the guinea-pig by using a battery of antibodies. In unfixed tissue, nearly all neurons in these ganglia demonstrated some degree of neurofilament protein triplet immunoreactivity. Large neurons generally displayed intense immunoreactivity, whereas most small to medium-sized neurons showed faint to moderate immunoreactivity. Double-labelling immunofluorescence demonstrated that most antibodies to the individual subunits of the neurofilament protein triplet had the same distribution and intensity of labelling in sensory neurons. Increasing durations of tissue fixation in aldehyde solutions selectively diminished neurofilament protein triplet immunoreactivity in small to medium-sized neurons. Double-labelling with neurofilament protein triplet antibodies in combination with antibodies to other neuronal markers, such as neuron-specific enolase, substance P and tyrosine hydroxylase, showed that tissue processing conditions affect the degree of co-localization of immunoreactivity to the neurofilament protein triplet and to these other neuronal markers. These results indicate that, with a judicious manipulation of the duration of tissue fixation, neurofilament protein triplet immunoreactivity can be used in combination with other neuronal markers to distinguish groups of neurons according to their size and chemical coding.

Animals↗
PubMed2004

An arthroscopic stitch for massive rotator cuff tears: the Mac stitch.

Arthroscopic repair of rotator cuff tears has become popular with the advancement in technology and arthroscopic technique. As we attempt to arthroscopically repair larger rotator cuff tears, we are relying more on tissue fixation. The tendon-suture interface has been recognized as the weak link in rotator cuff repair. In this article, we propose the use of the Mac stitch--a simple modification of suture placement, a combination of a horizontal and vertical loop at the site of repair--to increase the strength of tissue fixation. The Mac stitch is a simple arthroscopic stitch that can be used for small and massive rotator cuff repairs.

Arthroscopy↗
PubMed1999

Cold-temperature plastic resin embedding of liver for DNA- and RNA-based genotyping.

The standard practice of tissue fixation in 10% formalin followed by embedding in paraffin wax preserves cellular morphology at the expense of availability and quality of DNA and RNA. The negative effect on cellular constituents results from a combination of extensive cross-linking and strand scission of DNA, RNA, and proteins induced by formaldehyde as well as RNA loss secondary to ubiquitous RNase activity and negative effects of high temperature exposure during paraffin melting, microscopic section collection, and tissue adherence to glass slides. An effective strategy to correlate cellular phenotype with molecular genotype involves microdissection of tissue sections based on specific histopathological features followed by genotyping of minute representative samples for specific underlying molecular alterations. Currently, this approach is limited to short-length polymerase chain reaction amplification (<250 bp) of DNA, due to the negative effects of standard tissue fixation and processing. To overcome this obstacle and permit both cellular morphology and nucleic acid content to be preserved to the fullest extent, we instituted a system of cold-temperature plastic resin embedding based on the use of the water-miscible methyl methacrylate polymer known as Immunobed (Polysciences, Warminster, PA). The system is simple, easy to adapt to clinical practice, and cost-effective. Immunobed tissue sections demonstrate a cellular appearance equivalent or even superior to that of standard tissue sections. Moreover, thin sectioning (0.5-1.0 microm thickness) renders ultrastructural evaluation feasible on plastic-embedded blocks. Tissue microdissection is readily performed, yielding high levels of long DNA and RNA for genomic and transcription-based correlative molecular analysis. We recommend the use of Immunobed or similar products for use in molecular anatomical pathology.

Cold Temperature↗
PubMed2001

HOPE fixation: a novel fixing method and paraffin-embedding technique for human soft tissues.

We have developed a novel method for tissue fixation, including subsequent paraffin-embedding and sectioning, that allows the complete pathological analysis of all types of human soft tissues. Furthermore, it maintains additional positive features relevant to immunohistochemistry and molecular pathology. The so-called HOPE-technique (Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) comprises a protection-solution with an organic buffer, acetone as the only dehydrating agent, and pure paraffin of 52-54 degrees C melting temperature. Although the exact mechanism of protection has still to be elucidated, it seems rather unlikely that chemical bindings occur during the whole process of fixation, which is described and compared with the standard formalin-paraffin technique. Essentially, HOPE-fixed sections show formalin-like morphology. However, the sections are somewhat difficult to handle because of their fragility. This is due to the absence of any type of protein cross-linking and the dynamic processes of immersion and outflow of the HOPE protection solution. HOPE-fixed sections provide an excellent preservation of proteins and antigenic structures for differential analysis by immunohistochemical and/or enzyme histochemical techniques. However, their most remarkable feature is the extremely low degradation of nucleic acids (DNA and RNA) combined with good results obtained by in situ hybridization techniques. In conclusion, HOPE fixation may become a valuable additional tool in modern pathology.

Cross-Linking Reagents↗
PubMed1994

Oncogene expression in cervical cancer.

Significant advances have been made toward the understanding of the initiation and progression of cervical dysplasia and neoplasia at the molecular level. To date, this has not translated into improvements in diagnosis or treatment although it is a realistic expectation that this will occur. Significant variation in the proportion of tissue specimens that exhibit genetic alterations is striking. This may be attributed to different methods of analysis, different methods of tissue fixation, which influence antigen preservation, and the analysis of small numbers of samples per report, which introduces the possibility of sampling error. In spite of the variation among published reports, it is clear that several genetic alterations occur in preneoplastic and early-stage invasive cervical neoplasms. It remains to be determined which alterations of genetic structure or expression contribute to tumor initiation. The prognostic applicability of oncogene mutations is a particularly interesting area of investigation that is the closest to clinical application, although additional research involving larger numbers of patients is critical. The development of convenient methods of tissue fixation that preserve the myc oncoprotein, the synthesis of specific antibodies that provide consistent results, and the application of computer-assisted image analysis to quantitate results will be particularly important in this regard.

Female↗
PubMed1984

Polyester wax embedding and sectioning technique for immunohistochemistry.

We have developed a method useful for immunohistochemical studies by combining tissue fixation with buffered neutral formalin and polyester wax embedding. Buffered neutral formalin fixation preserves cell and tissue fine structure, and also the antigenicity of unstable enzymes. Polyester wax embedding makes possible thin serial sections of various tissues and preserves antigenicities for at least 6 months. We have demonstrated using this technique the localization of alpha-amylase in mouse salivary gland, parietal-cell specific antigen in mouse glandular stomach, and DNA polymerase alpha and beta in chick tissue.

Animals↗
PubMed2006

The effect of surgically induced ischaemia on gene expression in a colorectal cancer xenograft model.

Delays in tissue fixation following tumour vascular clamping and extirpation may adversely affect subsequent protein and mRNA analysis. This study investigated the effect of surgically induced ischaemia in a xenograft model of a colorectal cancer on the expression of a range of prognostic, predictive, and hypoxic markers, with a particular emphasis on thymidylate synthase. Vascular occlusion of human tumour xenografts by D-shaped metal clamps permitted defined periods of tumour ischaemia. Alterations in protein expression were measured by immunohistochemistry and spectral imaging, and changes in mRNA were measured by reverse transcriptase-polymerase chain reaction. Thymidylate synthase expression decreased following vascular occlusion, and this correlated with cyclin A expression. A similar reduction in dihydropyrimidine dehydrogenase was also seen. There were significant changes in the expression of several hypoxic markers, with carbonic anhydrase-9 showing the greatest response. Gene transcriptional levels were also noted to change following tumour clamping. In this xenograft model, surgically induced tumour ischaemia considerably altered the gene expression profiles of several prognostic and hypoxic markers, suggesting that the degree of tumour ischaemia should be minimised prior to tissue fixation.

Animals↗