A histological comparison of phase-partition fixation with fixation in aqueous solutions

In phase-partition fixation, tissue is immersed in a non-aqueous solvent at equilibrium with an aqueous solution of a fixing agent to minimize osmotic effects. Preservation of morphology afforded by phase-partition fixation using formalin and glutaraldehyde and several organic solvents was compared to aqueous 10% neutral buffered formalin fixation for five tissues. It was shown that phase-partition fixation can provide excellent fixation for light microscopy if the proper combinations of fixatives and solvents are used.     

Commercial Formalin Substitutes for Histopathology

We compared the performance of six commercial fixatives proposed to be formalin substitutes with the performance of buffered formalin, Clarke's ethanol-acetic acid, and ethanol, using rat liver, small intestine, and kidney. We investigated the rate of penetration, mode of fixation, extent of protein and structural immobilization, quality of histology and cellular structure following routine dehydration and paraffin embedding, and performance as a fixative for immunohistochemistry. Furthermore, we evaluated the effects of the various fixatives on ultrastructure. Only buffered formalin performed equally well on all tissues tested. While several of the commercial fixatives appeared to preserve liver tissue at 200, the preservation of kidney, intestinal villi, and smooth muscle was unacceptable. Histological distortion, cell shrinkage and vacuolization were prominent when the substitutes or ethanol were used. In contrast, these artifacts were found occasionally and to a minor degree when buffered formalin or Clarke's fixative were used. Immunohistochemistry demonstrated a total loss of low molecular weight antigen (serotonin) and patchy reactions for high molecular weight antigens for all fixatives except buffered formalin. The best immunostaining was obtained by combining formalin fixation with antigen retrieval. We conclude that none of the proposed commercial substitutes for buffered formalin are adequate for critical histology or histopathology.     

Effects of tissue processing techniques in acoustical (1.2 GHz) and light microscopy.

In this study the influence of various tissue processing and staining techniques on the acoustical properties of liver tissue was investigated. A qualitative study was performed using ultrasound attenuation as the imaged parameter of a combined optical/acoustical microscope with a 1.2 GHz transducer. Images were made of three sets of adjacent liver sections (6 microns in thickness) which were prepared in ten different ways: fixed by alcohol or formalin; stained by hematoxylin-eosin (HE), toluidine blue (TB) or non-stained; sectioned by a cryostat or by a paraffin microtome. It was concluded that the images obtained from cryostat sections were of much higher quality than those from paraffin sections. Images obtained from sections that were sectioned while embedded in paraffin displayed no detail at all. No consistent effect was noticed with respect to staining by HE or TB. Alcohol fixed sections gave more detailed images than formalin fixed sections. Formalin fixation in combination with cryostat sectioning yielded many cytoplasmic vacuoles.     

Determination of partition coefficient by the change of main phase transition

The molar partition coefficients of amphiphilic additives, e.g. local anesthetics, between the aqueous phase, the liquid crystal and the gel phase of lipid membrane can be determined based on a combination of phase transition data obtained at high and low concentrations of the lipid in aqueous phase. The data obtained at high lipid concentration allow to find the phase diagram lipid-additive in the aqueous environment. The combination of this diagram with data obtained at low lipid and additive concentrations provides direct information on the concentration of anesthetics in the lipid and thus allows the calculation of the partition coefficient.     

Further Studies on the Lyo and Desmo Components of Several Hydrolytic Enzymes and Their Histochemical Significance

This report describes additional studies of the lyo and desmo components of esterase, alkaline phosphatase, acid phosphatase, leucine aminopeptidase, and β-glucuronidase. The techniques used have already been reported (7). Enzyme diffusion occurs to different degrees in different fixatives, and varies somewhat with each enzyme. Loss of enzymatic activity during fixation occurs as a result of both inactivation due to the chemical reaction of the fixative with the enzymic protein, and diffusion of the lyo component into the fixative. The amount of diffusion into formalin can be reduced by the addition of salts, sucrose, or methocel. The pH of the aqueous medium significantly influences the removal of the lyo fraction from the tissue section. A striking similarity can be noted in the proportions of each fraction of enzyme present in the kidney of the rat, dog, and man. The procedure of fixation and paraffin embedding of tissue blocks does not wholly prevent the diffusion of the lyo component from the tissue sections when they are subsequently immersed in the aqueous incubation medium.     

A solvent partition method for microscale ganglioside purification

A simple and rapid method for the purification of gangliosides from the total lipid extract of plasma, cells, or tissue is described. The novel component of the method is the partition of the dried total lipid extract in the three-component solvent system consisting of diisopropyl ether, 1-butanol, and 50 mM aqueous NaCl (6/4/5, v/v/v). Gangliosides partition nearly quantitatively into the lower aqueous phase, and other lipids into the upper organic phase, resulting from the mixture of these three solvents. The ganglioside-containing aqueous phase is then freed of salts and other low-molecular-weight impurities by gel filtration. The thin-layer chromatographic patterns of total gangliosides thus obtained are clear and distinct, even when small samples with very low ganglioside concentrations (e.g., 1-ml samples of plasma) are processed by this method. Thus, this new ganglioside purification method is especially applicable to comparative qualitative studies of gangliosides requiring the analysis of multiple small samples.     

Simultaneous biosynthesis and purification of two extracellular Bacillus hydrolases in aqueous two-phase systems

Thermostable alpha-amylase with temperature optimum at 80 degrees C, molecular mass 58 kDa and pI point 6.9 was purified from a catabolite resistant Bacillus licheniformis strain. The enzyme was sensitive to inhibition by metal ions and N-bromosuccinimide. The partition behaviour of this enzyme in aqueous two-phase systems (ATPS) of the polymer-polymer-water type was investigated and some effects of type, molecular weight and concentration of phase components were studied. Up to 100% retention in the bottom phase of polyethylene glycol 10,000-20,000/dextran 200 system was reached. Best partition conditions were obtained in PEG 10,000-20,000/polyvinyl alcohol 200 systems, where the partition coefficient K increased 750 times to 7.5. Simultaneous production and purification of alpha-amylase and serine proteinase in PEG-polymer-water ATPS were examined. In the system PEG 6,000/ficoll, up to 90% of the amylase was retained in the bottom phase, whereas about 95% of the total protein (K = 22.8) and 60-75% of the proteinase were in the top phase. Similar separation of the enzymes from laboratory supernatant was obtained in system PEG/Na2SO4.     

Site-activity relationship of nitroxide radical's antioxidative effect

A relatively new strategy in preventing oxidative damage employs cyclic nitroxides. These stable radicals have been widely used as biophysical probes, spin labels, and are currently tested as contrast agents for nuclear magnetic resonance imaging. Nitroxides were found to protect cells, organs, and whole animals against diverse oxidative insults. The present study concentrated on comparing the antioxidative activity of nitroxides against oxidative damage, initiated either in the lipid or aqueous phase, to egg phosphatidylcholine acyl chains (13.4% polyunsaturated fatty acids) in small unilamellar vesicles. We determined the lipophilicity and liposome-membrane/aqueous-medium partition coefficient for several nitroxides and compared their specific protective effects. The aim was to study the relation between nitroxides' concentration, location in the lipid bilayer, and their protection against oxidative damage. Both 6-membered- and 5-membered-ring nitroxides were studied for: (i) partitioning between the lipid bilayer and the aqueous phase (nitroxides were quantified using EPR spectroscopy); (ii) the intrabilayer distribution, using three different fluorescent probes of known location of their fluorophors in the lipid bilayer; and (iii) the specific antioxidative effect (protection per concentration) against radicals formed in a liposomal dispersion. The radicals were generated using the thermolabile, radical-generating compounds 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) in the aqueous phase, and 2,2'-azobis (2,4-dimethyl-valeronitrile) (AMVN) in the lipid phase. The results show that nitroxides react, in a concentration-dependent manner, with deleterious species at their formation sites, both in the aqueous and the lipid phase, and that their specific protective effects for the lipophilic target, the lipid bilayer, are similar for both the lipophilic and the hydrophilic nitroxides.     

Staining of demineralized cartilage. I. Alcoholic versus aqueous demineralization at neutral and acidic pH

Demineralization of cartilage with alcoholic EDTA provides cartilage staining that is no better, as measured by scanning microdensitometry, than that of adequately fixed specimens demineralized with aqueous EDTA. Aqueous EDTA is a faster demineralizing agent than alcoholic EDTA. Certain fixatives can preserve maximal proteoglycan staining in articular cartilage even with subsequent rapid demineralization in formate buffer at pH 3.3. Although alcoholic formalin fixation provided optimum quantitative cartilage staining, cetylpyridinium chloride (CPC) in aqueous buffered formalin improved cellular detail, but CPC partially suppressed matrix staining.     

Effects of tissue processing techniques in acoustical (1.2 GHz) and light microscopy

Summary In this study the influence of various tissue processing and staining techniques on the acoustical properties of liver tissue was investigated. A qualitative study was performed using ultrasound attenuation as the imaged parameter of a combined optical/acoustical microscope with a 1.2 GHz transducer. Images were made of three sets of adjacent liver sections (6 m in thickness) which were prepared in ten different ways: fixed by alcohol or formalin; stained by hematoxylineosin (HE), toluidine blue (TB) or non-stained; sectioned by a cryostat or by a paraffin microtome. It was concluded that the images obtained from cryostat sections were of much higher quality than those from paraffin sections. Images obtained from sections that were sectioned while embedded in paraffin displayed no detail at all. No consistent effect was noticed with respect to staining by HE or TB. Alcohol fixed sections gave more detailed images than formalin fixed sections. Formalin fixation in combination with cryostat sectioning yielded many cytoplasmic vacuoles.     

Studies on Bovine Mast Cells: I. The Effect of Formalin Fixation

Studies are reported on the effect of aqueous formalin fixation on the bovine mast cell. Paraffin sections of bovine skin were prepared from tissues fixed for 7 to 72 hr in 10% neutral formalin in saline, and from tissues which were treated with water for various times both before and after formalin fixation. Metachromatic halos, scattering of the granules, and fraying of the cell outline, were seen in the mast cells of tissues washed in water before fixation. Exposure to water after fixation did not produce these artifacts. The tendency towards orthochromatic staining, and the occurrence of perinuclear clear zones are probably effects of the formalin and not of overstaining or of exposure of the tissue to water. The majority of the metachromatic material in the bovine mast cell is water soluble, and may be removed by washing in water before fixation, whereas the granules are relatively resistant to water. The optimum time of formalin fixation of bovine skin to permit studies on the metachromatic material of the mast cells was 24 to 36 hr.     

Aqueous two-phase polymer partitioning of lipid vesicles of defined size and composition

The kinetics of the partitioning of lipid vesicles containing acidic phospholipids in aqueous two-phase polymer systems are dependent upon the vesicle size; the larger the vesicles, the more readily they absorb to the interfaces between the two polymer phases and hence are cleared from the top phase as phase separation proceeds. The partitioning of neutral lipid vesicles is principally to the bulk interface and is the same in phase systems of both low and high electrostatic potential difference between the two phases (delta psi). The incorporation of negatively charged lipids has two effects upon partition. First, vesicles with negatively charged lipids exhibit increased bottom phase partitioning in phases of low delta psi due to an enhanced wetting of the charged lipids by the lower phase. Second, the presence of a negatively charged group on the vesicle surface results in increased partition to the interface and top phase in phase systems of high delta psi. Differences observed in the partition of vesicles containing various species of negatively charged lipid thus reflect a competition between these two opposing factors.     

The partition of cis-parinaric acid and trans-parinaric acid among aqueous,fluid lipid,and solid lipid phases

Summary In this article, I review the current information concerning the partition of the fluorescent probes, cis-parinaric acid (9, 11, 13, 15-cis, trans, trans, cis-octadecatetraenoic acid) and trans-parinaric acid (9, 11, 13, 15-all trans-octadecatetraenoic acid) among aqueous, solid lipid, and fluid lipid phases. The association of these probes with lipid is described by a mole fraction partition coefficient whose value is typically in the range of 1–5 × 10⁶, a reasonable value in light of partition coefficients for other fatty acids between hydrophobic phases and water. The partition coefficient, in the absence of lipid phase changes, is relatively independent of temperature and only slightly dependent on the total aqueous probe concentration.In lipid samples which contain coexisting fluid and solid phases, trans-parinaric acid preferentially partitions into the solid phase, while cis-parinaric acid distributes nearly equally between fluid and solid phases. This partition behavior probably arises from the molecular shape of the cis and trans parinaric acid isomers. From measurements of the polarization of fluorescence of cis and trans parinaric acid in mixed lipid systems or membranes it is possible to evaluate the proportion of lipid components involved in phase changes or phase separation. From fluorescence energy transfer between protein typtophan residues and the parinaric acid isomers it is possible to gain information about the organization of lipids and proteins in membranes and model systems. I close the review by considering some of the membrane research areas where these probes and their various lipid derivatives may be particularly useful.     

Kinetic theory of enzymatic reactions in reversed micellar systems. Application of the pseudophase approach for partitioning substrates

A kinetic theory is proposed for enzymatic reactions proceeding in reversed micellar systems in organic solvents, and involving substrates capable of partitioning among all pseudophases of the micellar system i.e. aqueous cores of reversed micelles, micellar membranes and organic solvent. The theory permits determination of true (i.e. with reference to the aqueous phase, where solubilized enzyme is localized) catalytic parameters of the enzyme, provided partition coefficients of the substrate between different phases are known. The validity of the kinetic theory was verified by the example of oxidation of aliphatic alcohols catalyzed by horse liver alcohol dehydrogenase in the system of reversed sodium bis(2-ethylhexyl)sulfosuccinate (AOT, aerosol OT) micelles in octane. In order to determine partition coefficients of alcohols between phases of the micellar system, flow microcalorimetry technique was used. It was shown that in the first approximation, the partition coefficient of the substrate in a simple biphasic system consisting of water and corresponding organic solvent can be used as an estimate for the partition coefficient of the substrate between aqueous and organic solvent phases of the micellar system. True values of the Michaelis constant of alcohols in the micellar system, determined using suggested approach, are equal to those obtained in aqueous solution and differ from apparent values referred to the total volume of the system. The results clearly show that the previously reported shift in the substrate specificity of HLADH, observed on changing from aqueous solution to the system of reversed aerosol OT micelles in octane, is apparent and can be explained on the basis of partitioning effects of alcoholic substrates between phases of the micellar system.     

Effects of Fixation and Tissue Processing on Immunohistochemical Demonstration of Specific Antigens

Identification of biomarkers in archival tissues using immunochemistry is becoming increasingly important for determining the diagnosis and prognosis of tumors, for characterizing preinvasive neoplastic changes in glandular tissues such as prostate, for evaluating the response of tumors and preinvasive neoplastic changes to certain therapies (i.e., as a surrogate intermediate end point), for selecting patients who are candidates for specific therapies (e.g., immunotherapy) and for retrospective studies. For detecting specific biomarkers it is important to understand the limitations imposed by the fixation methods and processing of the tissues. This study was designed to determine the effects of fixation on the detection in archival paraffin blocks of selected antigens postulated to be important in tumor biology. We evaluated the antigens TGFα, p185^erbB-2, broad spectrum keratins, p53, and TAG-72 (B72.3). Fixatives evaluated included standard preparations of neutral buffered formalin, acid formalin, zinc formalin, alcoholic formalin, ethanol, methanol, and Bouin's fixative. We found that in general neutral buffered formalin is the poorest fixative for maintaining antigen recognition by immunohistochemistry and that no single fixative was best for all antigens. The dehydrating (coagulant) fixatives (e.g., ethanol and methanol) preserved immunorecognition of p53 and broad spectrum keratins best while the slow cross-linking fixatives (e.g., unbuffered zinc formalin) were best for demonstrating TGFα and p185^erbB-2. Fixatives other than neutral buffered formalin produced equivalent recognition of the epitope of TAG-72 by B72.3. In formalin fixed archival tissues, only a portion of the antigen signal can be detected by routine immunohistologic methods.     

Detection of albumin mRNAs in rat liver by in situ hybridization: usefulness of paraffin embedding and comparison of various fixation procedures

Our aim was to define optimal conditions for efficient and reproducible albumin mRNA detection in rat liver by in situ hybridization. We used an albumin-specific [3H]-labeled cDNA probe with a specific activity of 6-8.10(6) cpm/microgram DNA. In situ hybridization is as efficient on paraffin sections as on cryostat sections for detecting albumin mRNAs. Perfusion fixation with a 4% paraformaldehyde solution results in homogeneous RNA retention within tissue blocks, in contrast with immersion fixation, which yields heterogeneous RNA preservation. Comparison of immersion fixation with three different fixatives (paraformaldehyde, ethanol-acetic acid, and Bouin's fixative) shows that the highest level of hybridization signal is obtained with paraformaldehyde. Ethanol-acetic acid and Bouin's fixative appear less efficient for albumin mRNA detection. Loss of mRNAs within liver tissue blocks over time is largely although not completely prevented by paraffin embedding.     

Fixation with even small quantities of glutaraldehyde affects red blood cell surface properties in a cell- and species-dependent manner. Studies by cell partitioning

Partitioning in charge-sensitive dextran-poly(ethylene glycol) aqueous phase systems reveals that fixation with even small concentrations of glutaraldehyde (e.g., 0.1% w/v) changes the surface properties of cells. While fixation with larger concentrations of glutaraldehyde (i.e., 1.85%) increases erythrocyte partition ratios, the effect of lower glutaraldehyde concentrations on the partition ratios appears to be species-specific. The differential effect of glutaraldehyde on rat reticulocytes and erythrocytes indicates that fixation is also cell-dependent. These data, together with the previous report that glutaraldehyde fixation does not change the characteristicrelative partition ratios of rat mature erythrocytes of different cell ages, suggest that the nature and extent of glutaraldehyde alteration of cell surfaces must, in each case, be empirically evaluated.     

Simultaneous biosynthesis and purification of two extracellular Bacillus hydrolases in aqueous two-phase systems

Thermostable a-amylase with temperature optimum at 80 °C, molecular mass 58 kDa and pI point 6.9 was purified from a catabolite resistant Bacillus licheniformis strain. The enzyme was sensitive to inhibition by metal ions and N-bromosuccinimide. The partition behaviour of this enzyme in aqueous two-phase systems (ATPS) of the polymer-polymer-water type was investigated and some effects of type, molecular weight and concentration of phase components were studied. Up to 100% retention in the bottom phase of polyethylene glycol 10,000—20,000/dextran 200 system was reached. Best partition conditions were obtained in PEG 10,000—20,000/polyvinyl alcohol 200 systems, where the partition coefficient K increased 750 times to 7.5. Simultaneous production and purification of a-amylase and serine proteinase in PEG-polymer-water ATPS were examined. In the system PEG 6,000/ficoll, up to 90% of the amylase was retained in the bottom phase, whereas about 95% of the total protein (K = 22.8) and 60—75% of the proteinase were in the top phase. Similar separation of the enzymes from laboratory supernatant was obtained in system PEG/Na₂SO₄.     

Special symposium: fixation and tissue processing models

Abstract

Fixation and processing of tissue to paraffin blocks permit thin (4-5 µm) sections of tissues to be cut. Tissues and their subcellular components and surrounding stroma are visualized by cutting thin sections and staining them histochemically or immunohistochemically and viewing the sections using a bright field microscope. During the last century, anatomists and pathologists have used fixation with 10% neutral buffered formalin (10% NBF) as the fixative of choice. Also, both human and veterinary pathologists have trained to use fixation with 10% NBF, so these professionals are reluctant to change the familiar microscopic appearance of diagnostic tissues by using different fixatives. In addition, the effects of tissue processing on the microscopic appearance of tissue essentially has been ignored in most studies. Archives of paraffin blocks of pathological tissue contain essentially paraffin blocks fixed in 10% NBF. Therefore, if retrospective studies use archival paraffin blocks to correlate the molecular features of diseases with their outcomes, the studies must be based on tissue fixed in 10% NBF. Studies of how fixation in 10% NBF interacts with histochemical and immunohistochemical staining are limited in number and most are based on relatively long fixation times (≥36 h). Currently, fixation times in 10% NBF have been reduced to <24 h. Little is known about fixation in 10% NBF and its interaction with tissue processing for any period of fixation, especially short times. Less is known about how fixation of tissues with 10% NBF interacts with more modern assays using immunohistochemistry, real time quantitative polymerise chain reaction (PCR), and techniques that depend on analysis of proteins extracted from paraffin blocks including multiplex immunoassays or mass spectrometry. In general, multiple antibody–antigen combinations are reported not to work in tissues fixed in 10% NBF, i.e., loss of immunorecognition is nearly complete for such antibody–antigen combinations as Ki67/MIB, estrogen receptor alpha (ERα) and Progesterone receptor (PR), and partial for Bcl-2. Several models have been developed to study the interactions of tissue fixation and immunorecognition, but most have viewed the problem with immunorecognition as completely caused by fixation. Also, some of the models discussed in this special symposium do not predict the effects of fixation on frozen tissues fixed in 10% NBF and not processed to paraffin blocks. This article is a brief review of issues attending the use of 10% NBF combined with tissue processing as an interrelated process to study biomarkers identified by immunohistochemistry.