HIAR in frozen sections prepared from specimens fixed with glutaraldehyde or glutaraldehyde and osmium tetroxide.
\r\n\tNowadays, environmental contamination with heavy metals is one of the most severe environmental issues, which needs the synergistic action of the scientific community, the general public and diverse authorities. Unfortunately, industrial effluents containing hazardous residues are frequently discharged into the ecosystem, thus resulting in harmful effects to the environment. One of the principal sources of heavy metal pollution near rivers and estuarine streams is the intensified industrial use of metals and the process of leaching of mine tailings, hence, the drainage of untreated mines. The infiltration of heavy metals into watercourses is generally accompanied by sediment deposits that result in a high concentration of heavy metals in water, sediments and aquatic organisms. In various developing and emerging countries, studies have shown that agricultural land irrigated with wastewater contributes about 50% of plant resources to urban areas, hence, is of utmost importance for food security. Based on economic considerations, farmers are often forced to maximize production in order to generate profit, regardless of environmental concerns or human health.
\r\n\tConventional decontamination technologies used to clean up contaminated metal sites are relatively insensitive, costly and time-consuming, often dangerous for workers, and produce secondary waste, which in turn displays another environmental threat. Innovative and sustainable techniques could be promising solutions to remediation of heavy metal contamination.
Unerupted or impacted permanent tooth can involve any tooth in the dental arch. According to many authors, the teeth that are usually involved are the maxillary and mandibular third molars, the maxillary canines, and the mandibular second premolars, respectively. The delayed eruption of second permanent molars is an infrequent occurrence; however, it has clinical impacts when it occurs.
The frequency of occurrence in the impacted second molars is rare and can vary within 0% to 2.3% [1, 2, 3]. Permanent molars are exceptionally crucial for the dentition to develop normally and for the facial growth synchronization, along with providing occlusal support for masticatory functions. Any disturbance in the eruption path of permanent molars can lead in a short lower facial height [4].
Most of the time, the pathology from the impacted second molar may provoke many pathologic disorders to the adjacent and opposite teeth. Caries, periodontitis, roots resorption, or pericoronitis may occurred to those neighboring teeth. Other results may form the follicular cyst, tilting of neighboring teeth, and eventually malocclusions [5, 6].
The treatment modalities for unerupted second molars depend on the impaction’s severity and the stage of development of the patient [5], which often requires a multidisciplinary approach. The treatment options include surgical procedures [7, 8], orthodontic methods [9, 10], and combined surgical and orthodontic [11]. Some authors suggested extracting the unerupted molar and following orthodontic approaches [10, 12, 13]. Each method has its advantages and disadvantages.
Timely early diagnosis and treatment of permanent second molars’ impaction contribute to the most satisfactory consequences with promising and lasting prognosis [14]. Later treatment will be more problematic because not only will the clinical divergence increases with time, but also, the ability to adjust the existing dentition is less.
There are various treatment modalities of the second molar impactions. However, there are no clear guidelines for the clinician to follow when dealt with such cases. Therefore, this review will explain the etiology and incidence of impacted second permanent molars, emphasize the need for earlier diagnosis, and focus on treatment options that can guide the decision-making process.
The eruption of permanent molars diverges on or after the eruption of other permanent teeth because the former primary teeth are absent. The formation of permanent molars is consecutively originated in the maxillary tuberosity and at the connection of the mandibular ramus and body of the mandible. The jaws’ development results in the respective position of the first permanent molar to shift forward at the time of the development of the second molar. The occlusal surface of the mandibular molars is mesially inclined while the occlusal surface of maxillary molars is distally inclined during the beginning of the formation. The crowns then slowly shift to a more vertical position. Notably, there is an inherent close association between the eruption of a tooth and the stage of root development. Moreover, three-quarters of the roots of the tooth will be formed right after emergence. Remarkably, half of the roots of central incisors and lower first permanent molars have been developed by this time [5].
The concept of eruption failure is different from the delayed eruption. It is reflected as the incapacity of the tooth to erupt in the oral cavity [15]. This may occur in one or more teeth, with primary or the permanent dentition, and could possibly be incomplete and complete eruption failure. Bone or soft tissue may cover the teeth. In more ways, the failure is reliant on the pre-existing etiology [16].
A disruption in the eruption path means the second molar erupts into contact apical to the eminence on the first permanent molar’s distal surface, and from this occurrence, the second molar can be locked [17] (Figure 1). Impaction, by definition, is tooth retention owing to an obstruction in the tooth germ’s eruption path or ectopic position in the dentition [11]. Impaction is deemed to be the termination of the eruption of a tooth. The obstruction can be detected clinically or radiographically in the route of eruption or due to the tooth’s malposition. Primary retention stops the eruption of a customarily situated and developed tooth before the emergence, without a visible obstruction in the eruption path. Secondary retention is the ending of the tooth eruption after the emergence devoid of a physical barrier [2, 5, 18]. There are distinct treatment approaches for the eruption disturbances [5].
The impacted second molar that occur (arrow) A: a part of panoramic radiograph on the left side B: in the oral cavity C: the occlusion of the lower impacted second molar and upper second molar.
The diagnosis of impacted second molars has been made typically between 10 and 14 years of age. Helm and Seidler defined ordinary emergence for the second molar at 12.4 and 11.9 years in the maxilla and 11.9 and 11.4 years in the mandible for boys and girls, respectively [19]. The late emergence of the second molars is seldom the primary reason for an orthodontic recommendation [20].
The frequency of impacted second molars is only minimal and it varies from 0% to 2.3% [2]. Brondemark and Tsiopa found 2.3% of the wide-ranging prevalence of eruption disturbances of the permanent second molar. Cho et al. reported that the impaction mandibular second molars in Chinese children was only 1% [21]. The impacted second molars are usually overlooked whenever there is clinical examination, and radiographs might not be routinely taken for all cases. Furthermore, these impacted the second molar teeth are ignored by asymptomatic patients [22].
However, a greater incidence of second molar impaction is more common in orthodontic patients [23]. The prevalence of impacted second mandibular molar from orthodontic records of young caucasian was relatively high (1.36%) as the study was conducted on the orthodontic population [24]. The age of the individuals with impacted second molar varies from 9 to 26 years [1, 2]. Second molar impaction often ensues much more in the mandible than in the maxilla, which may arise for the reason that there is a later development of the upper third molar [25]. These impactions tend to be more unilateral than bilateral [2, 25]. Second molar impaction and retention are habitually detected during orthodontic treatment as a supplemental finding and are infrequently the main reason for referral to an orthodontic clinic [26].
Many factors are elaborated with the failure of eruption, affecting prognosis and treatment [20]. The impacted second molar is an asymptomatic pathology. Failure of eruption is also more likely to be a secondary finding during orthodontic treatment [27]. Andreasen et al. [4] concluded that three leading factors are drawn in the eruption interference. These causes incorporate position of the ectopic tooth, barriers in the eruption path, and failures in mechanism of the eruption [17, 28]. Posterior crowding is thought to be the most common cause of mesially angulated lower-second-molar impactions [11].
Heredity is also brought up as a secondary etiological factor [29]. Patients with specific syndromes may involve some systemic factors [16]. In the patients with a disturbance of local eruption, only one or a few teeth may be affected. The early diagnosis of eruption disturbances is crucial. It is essential to provide treatment at a suitable time and minimize complications [5, 25, 30].
An ectopic eruption of the maxillary first molars impaction is often related to a mesial angle path to the expected direction of eruption. It may also affect the second molar’s failure to upright from its mesial inclination during the emergence. Supernumerary teeth and odontogenic tumors or cysts can be the major physical barriers of the eruption path [5].
The eruption of mandibular second premolar and second molar usually in competition for space of the dental arch. When this posterior space in the arch is inadequate, the earlier emergence second premolar may cause the failure of eruption of the second molar [31].
The impacted second molars are diagnosed between 10 and 14 years of age [32]. Rapid diagnosis is crucial in improving prognosis and alleviating the effect of the failure of eruption. It requires a full clinical and radiographic examinations including previous medical record, which are enough to differentiate between impaction, primary, and secondary retention [5, 15].
As the time of eruption may differ among each child, follow-up at six-month intervals of children with mixed dentition is recommended to guide the pattern of eruption and the development of the teeth. The posterior crowding cases and the uncertain of the molar retention should be more concerned [21]. The orthodontist should be alerted if one erupted lower second molar without the contralateral molar is observed in the clinic. The number of teeth with delayed eruption compared to the contralateral teeth should be one of the cautions.
In a preadolescent patient, a panoramic radiograph showing a lower-third-molar follicle positioned on top of the developing second molar crown may give early warning of future impaction [32].
If the eruption of a permanent mandibular second molar is six-month delayed compared with its contralateral molar or both molars show a year delay in eruption, the dubitation of molar retention must necessitate further radiographic investigation [21]. The cone-beam computed tomography is a tool that can be employed to identify ankylosis [28].
Unerupted molar may be found in a panoramic radiograph thru a regular dental check-up or orthodontic assessment and treatment planning [20]. The radiological examination must focus on the follicles of unerupted molars, dental abnormalities, root abnormalities, dilacerations, taurodontism, invagination, resorption, or dental caries in both neighboring primary molar and first permanent molar [30].
The treatment option depends on the mandibular second molar impaction dictated by the degree of mesial angulation and its vertical depth, which could be seen in a panoramic radiograph. The inclination of impacted teeth is calculated from the angle between the first and second lower molars’ long axis. The impacted second molar is in mesial inclination if the angle between their long axis is more than 40°. If the angle of the long axis of both molars is between 40° and − 20°, the impacted second molar is in a vertical position, and if the angle is less than −20° means the impacted second molar is in distal inclination [20]. The vertical depth of impacted teeth will determine by the distance between the distal marginal ridge of the first molar to the mesial marginal ridge of the impacted second molar [22].
The treatment modalities for the second molar impaction depend on the variety of abnormalities of the eruption and the patient’s age. Treatment options may incorporate observation, repositioning or surgical exposure, orthodontic uprighting, and the extraction of unerupted molar. Each modality has its indications, contraindications, advantages, and disadvantages [20].
The orthodontic or surgical approach is indicated only when the etiologies are from impacted ectopic erupting teeth and primary retention [17]. However, a prior observation time is essential to confirm the diagnosis through a radiographic follow-up before any intervention. The natural eruption may occur in normal occlusion in a few cases [11].
The intervention should be deliberated after an observation period of 12 months when the possibility of self-correction is ruled out [21]. Abnormally positioned tooth germ of the third molar may create a physical barrier that causes the second molar impaction. The suggested treatment is removing the third molar at the 11–14 years age old together with a in-depth follow-up for the second molar [5].
Once there is no chance for self-correction, the parents and patients should be informed about the treatment option for the impacted molars. The treatment options may include orthodontic uprighting, surgical repositioning, the impacted second molar extraction and letting the third molar drift into the second molar position, and transplanting the third molar into the extraction site of the impacted second molar [21].
Diagnosis made early on and prompt treatment are vital to an effective treatment of mandibular second molar impaction. The orthodontic treatment is recommended for impacted or ectopically erupted teeth and in cases of primary retention [17]. Although orthodontic treatment is usually given in the mandibular arch the success rate is the same for both the arches [11].
The individuals between 11 and 14 years in which the roots of second molars are still incomplete are suggested to treat the impaction. The poor prognosis is the impacted molars with fully formed roots [5]. The success of orthodontic treatment relates to numerous local considerations such as the impacted tooth ‘s angulation, the third molar position, and the severity of crowding or follicle collision [11]. There are several methods to treat the second molar impaction. However, there are some limitations, specifically in the treatment of severely deep impacted teeth.
At the first stage of conventional orthodontic treatment, during the alignment and leveling, a tube is bonded to the molar’s buccal surface. It will be engaged with a continuous archwire. Super-elastic archwire and a push coil spring will ensure the alignment and distalization. The super-elastic archwire used for alignment and leveling of the teeth is curved distally of the impacted second molar. It is inserted in the tube to help uprighting the impacted second molar [31].
The techniques of uprighting the impacted mandibular second molar with a cantilever spring are optional treatments besides extraction or surgical repositioning of the tooth. The treatment can be directed with or without the removal of the adjacent third molar. It needs surgical exposure of the crown of the impacted second molar, followed by bonding an orthodontic attachment for uprighting the impacted tooth [33].
It is a straight forward way to upright the angulated and impacted second molar using an uprighting cantilever spring attached to a bonded tube on the distobuccal cusps of the affected tooth [31].
The cantilever mechanics designed was useful for the correction of an extremely tipped and deeply impacted molar. When the cantilever mechanics is used, the tipped molar will have an uprighting moment and an extrusive force.
Thus, an occlusal interference with the opposing tooth may occur. Morita et al. suggested utilizing a compression force with a two-step bend incorporated into a NiTi archwire for cases in which the molar was slightly tipped and extruded [34].
Several orthodontic techniques utilize the distalized segmented archwire to upright the impacted molar. Before the bonding of fixed orthodontic appliances, a segmented wire is engaged between the impacted second molar and the adjacent first molar. The segmented archwire for the technique must have super elasticity characteristics. The wire will be bent and bonded to the occlusal surface of the first permanent molar [35].
Bach technique is one of the non-surgical technique for uprighting mesially impacted mandibular molars. The technique used an .014“ x .025” Copper NiTi wire.36 Whereas other developed a technique using .016“ x .016” NiTi wire [36]. Fu et al. developed a polearm using 0.016”x 0.022″ titanium molybdenum alloy wire [22]. A major advantage of these techniques was that they could be utilized on both bonded and unbonded mandibular arch.
Temporary skeletal anchorages devices have some superior advantages. They could provide vertical and distal traction forces at the same time with a good moment and line of action. They could also diminish the side effects related with dental anchorage. There are two methods of utilizing a temporary anchorage device for uprighting molar impaction, direct and indirect anchorage.
Direct anchorage is when the teeth moved directly towards or against the mini-screw. Chang et al., reported the use of a ramus screw to upright the complex impacted second molar [37]. An indirect anchorage refers to stabilizing certain teeth via a rigid connection with the mini-screw and subsequent use of these stabilized anchors to move other teeth in the dental arch [38].
Because these impacted teeth have limited access, surgical approaches should be considered to help with their necessary uprighting. Techniques are as follows:
Surgical exposure of the buccal surface bonded with a bracket has been done by Going and Reyes- Louise in 1999 for an impacted second molar. The exposure resulted in the successful positioning of the second molar of 40 patients. They applied this technique for seven years with an acceptable prognosis [39].
Usually, the buccal surface will be exposed via soft tissue removal or drilling into the bone covering the tooth. Care must be given to drilling into the bone to not traverse through the second molar’s cementoenamel junction and root areas [39]. This method was discussed as the most successful treatment by Magnusson and Kjellberg. These researchers conducted a clinical trial involving 87 patients ranging from 11 to 19 years old. The study gained 70% favorable results, making it a recommended modality [11].
As mentioned above, the procedure to expose the second molar is to remove the buccal bone to gain the tooth’s surface visibility. In patients where, orthodontic treatment is not an option or contraindicated, luxation is the next step. Using a straight elevator, the second molar is gently and slightly manipulated to the tooth’s position, luxating occlusally and distally, reaching the approximate level of the occlusal plane.
Kravitz et al. claimed that this technique is suitable for conserving the apical blood supply since the tooth will remain within the socket [32]. Hence, the tooth can have a favorable prognosis, especially when there is still incomplete root formation [32, 40]. Disadvantages would be fractures and pulp necrosis due to the manipulation employed in this method [20].
To achieve the upright position of the impacted second molar, the extraction of the third molar might be necessary. The removal of the third molar can be done with a standard approach in these cases [41, 42, 43]. According to McAboy et al., a trough is made on the second molar’s distal to compensate for the distal movement. Two hands are recommended in doing this procedure: one hand on the elevator to change the position of the tooth occlusal and distally, less than 75 degrees, five while the other hand will be for the cortical plate and alveolar ridge support [41].
The surgeon was said to feel a “snap” into the trough provided in the distal area, possibly indicating a stable position [44]. However, the tooth was advised to be placed off-occlusion slightly to avoid trauma on the site, and necessary stabilizing techniques can be performed if there is mobility [41]. This technique was widely evidenced to have a good prognosis and claimed to have no unfavorable sequelae in long-term follow-ups [43, 44]. Furthermore, other authors supported that removing the third molar might not be required in these cases [24, 40].
Autogenous bone grafting or any bone substitutes can be applied in this technique for stabilization in some cases where there are areas devoid of bone [41, 44]. On the other hand, Boynton and Lieblich suggested that bone grafts are not necessary for the stabilization process [45].
Subsequently, suppose all other minimally to moderately invasive surgical modalities fail. In that case, the prognosis is poor, and pathology has commenced— then extraction can be an option [33]. This method was said to be the most unsuccessful among all the modalities tested by Magnusson and Kjellberg since it was reported that the third molars that replaced these teeth resulted in a mispositioned state. Once this treatment is indicated, proper patient education on untoward outcomes is suggested [11].
Immediate transplantation of the third molar into the intentionally extracted second molar site is also an indicated management. Still, it possesses immense risk as there will be periodontal and pulpal complications after the procedure [32, 41]. Successful outcomes depend on the generation of new periodontal ligament and cellular structures, suggesting that the tooth should be transplanted right away to make way for a better prognosis.
According to Tsukiboshi, this treatment’s indications include the donor and recipient teeth should be morphologically analogous to each other; both teeth should be prepared with less invasive procedures; if endodontic treatment is deemed necessary, the therapy completion should be completed after two weeks [46]. In this procedure, failure happens when there is a pulpal infection, ankylosis, and resorption on the transplanted tooth [47]. The use of a piezosurgery machine in autotransplantation has been developed and resulted in an effective modality in harvesting the third molar to replace a second molar [48].
Preferably, the literature suggests that surgical repositioning approaches should be made in a second molar with half or two-thirds of root formation. Dessner [49] described that there is minimal root fracture that can happen in this development stage. Intervention in the earlier stages of root formation rather than the recommended one results in a displaced, unstable, and less than the second molar’s desired position.
Second molar impaction and retention are infrequently the initial reason for orthodontic treatment recommendation because they are rare and asymptomatic. It may be discovered in a regular panoramic radiograph during routine orthodontic evaluation. The diagnosis and treatment planning, which should consider the patient’s age is crucial. The treatment options include observation, surgical extraction of unerupted permanent molars, and a number of orthodontic and surgical methodologies for purposely uprighting the impacted molars. Though there are no definite guidelines for managing the second molar impaction, this literature can assist in the decision-making process and treatment planning of such clinical cases.
The authors would like to thank the staff, colleagues, and dental assistants including co-workers in Walailak University International College of Dentistry. Finally, the authors would like to thank Christian Estacio from Walailak University International College of Dentistry for editing and revising the language of this manuscript.
The authors declared the conflict of interest.
Conceptualization: Irin Sirisoontorn, methodology: Irin Sirisoontorn, validation: Irin Sirisoontorn, formal analysis: Irin Sirisoontorn, investigation: Irin Sirisoontorn, resources: Dinesh Rokaya, Bishwa Prakash Bhattarai, data curation: Irin Sirisoontorn, writing—original draft preparation: Ann Chianchitlert, Diane Selvido, writing—review, and editing: Irin Sirisoontorn, visualization: Irin Sirisoontorn, supervision: Natthamet Wongsirichat, final approval: Irin Sirisoontorn.
This study did not receive funding by a research grant or any sponsorship.
This study did not require ethical approval because it is a review literature.
There is no need for patient consent.
Immunohistochemistry is used for identifying the localization of cellular or tissue constituents (antigens) based on antigen-antibody interactions using labeled antibodies that can be visualized under light and electron microscopes. Therefore, the use of specific primary antibodies and tissue preparation techniques that conserve fine structures, the immobilization of antigens, and antigen-antibody interaction is essential. Until the end of the 1980s, the conservation of protein conformation was thought to be important for the immunohistochemistry of protein antigens for the following reasons. (1) The production and specificity of antibodies were mainly confirmed by immunoprecipitation using Ouchterlony double diffusion test and immunoelectrophoresis, in which native purified antigen proteins or tissue extracts were reacted with antisera to form precipitation lines at the proper antigen/antibody ratio in agar or agarose gels. (2) Strong immunoreactions were observed in tissues and cells that were fixed using formaldehyde within a short time or using other physical fixatives, such as cold acetone. (3) Frozen sections provided stronger immunostaining than paraffin sections, which denatured protein conformations during dehydration and embedding.
On the other hand, monoclonal antibodies began to be prepared in many laboratories in the 1980s, and commercially available monoclonal antibodies that recognized special cell types were being applied for pathological diagnosis. Western blotting and enzyme-linked immunosorbent assays (ELISAs) were introduced as the main techniques for detecting antigen-antibody reactions. Since these techniques involve the immobilization of both native and denatured antigens on membranes or plastic plates and antigen-antibody reactions are visualized using enzyme-labeled antibodies, these techniques have a higher sensitivity than immunoprecipitation methods independent of the antigen/antibody ratio. These new antibody preparation techniques and assay systems have gradually changed the concept that it is important to expose epitopes for immunoreactions rather than preserving the conformation of whole antigen molecules.
Histopathologists have made great efforts to use formalin-fixed and paraffin-embedded (FFPE) specimens for immunohistochemistry, since such specimens have long been used as the standard for light microscopy and are archived in many biological and clinical laboratories. In the early 1970s, treatments with enzymes such as proteases, nucleases, and hyaluronidase and with protein denaturants were introduced to enable the partial recovery of immunostaining. The development of heat-induced antigen retrieval (HIAR), as reported by Shi et al. in 1991 for FFPE specimens, completely changed the concept of immunohistochemistry [1]. Although the mechanism of HIAR was originally a mystery, several studies have elucidated that heating cleaves chemical crosslinks (methylene bridges) formed by formaldehyde and exposes epitopes in tissues [2, 3]. HIAR is now applied not only to FFPE sections but also to frozen sections, cultured cells, physically fixed materials, and plastic embedded specimens for both light and electron microscopy [4]. It is also applied to other histochemical fields, such as in situ hybridization and lectin histochemistry. Furthermore, FFPE specimens are recognized as useful resources to study protein expression, DNA aberrations, and RNA expression in normal and diseased tissues [5, 6].
In this chapter, the following topics will be described, focusing on a flexible reconsideration of the concept of immunohistochemistry: (1) antigen-antibody interactions in tissues, (2) mechanisms of chemical fixation, (3) mechanisms of HIAR in formaldehyde-fixed specimens including exposure of highly masked epitopes, (4) HIAR in immunoelectron microscopy including the use of conventionally processed specimens embedded in epoxy resins, and (5) effects of antibody diluents on immunohistochemistry.
Antibodies recognize epitopes, which are small areas of antigen proteins but not the entire antigen molecule. When purified proteins from tissues or recombinant proteins are injected into animals, polyclonal antibodies that react with multiple epitopes and have different affinities and immunoglobulin classes for each epitope are generated. If a synthesized linear peptide is used for the immunogens, generated antibodies with different affinities and classes and subclasses of immunoglobulins may react with a single epitope. Therefore, we should notice that a commercially available polyclonal antibody may show different immunoreactions when we use the antibody with different lot numbers. On the other hand, a monoclonal antibody reacts with a single epitope with a monovalent affinity and a single immunoglobulin class and subclass. Antibodies prepared using the phage display technique have almost the same characteristics as monoclonal antibodies.
There are two types of epitopes, i.e., linear and conformational epitopes [4]. Linear epitopes are composed of a particular stretch of 5–20 consecutive amino acids. Linear epitopes located on the surfaces of protein molecules can react with antibodies independent of whether the protein is in its native or denatured and extended states. Meanwhile, linear epitopes located on surfaces in contact with other proteins or the subunits and internal linear epitopes cannot bind to antibodies when the protein is in its native state and can only bind to antibodies when the polypeptides have been extended by heating or treatment with protein denaturants, such as sodium dodecyl sulfate (SDS) and urea. A few linear internal epitopes are rarely in contact with antibodies because the antigen proteins contain multiple disulfide bonds and the protein remains stable even after heating. The reduction of these disulfide bonds may be necessary for the exposure of such epitopes, as described later. Conformational epitopes are composed of amino acids that are located far apart in their linear sequence but become juxtaposed when the protein is folded into its native shape. Some conformational epitopes are stabilized by disulfide bonds and resistant to denaturation by heating and protein denaturants, but others associated with noncovalent forces may be sensitive to denaturation.
In immunohistochemistry, fixation is essential to conserve tissue structures and to prevent the diffusion of antigens. Chemical fixatives directly modify epitopes and crosslink proteins and nucleic acids to form gel-like structures that inhibit antigen-antibody interactions. The antigen-antibody reactions can be interfered with in special regions, such as cell organelles with intact membrane and secretory granules and deposits of protein fibrils that contain highly concentrated proteins, as well as in nuclei and extracellular matrices with highly negative electrostatic charges. Therefore, immunohistochemistry must be performed under conditions that promote the epitope-antibody association in the target tissues through the use of tissue-processing procedures, i.e., fixation, embedding, antigen retrieval, and the selection of suitable diluents for antibodies.
Chemical fixatives used for immunohistochemistry are limited to formaldehyde and glutaraldehyde. Formaldehyde is used for tissue fixation in both light and electron microscopy, while glutaraldehyde is used as a fixative only in electron microscopy. Although formaldehyde and glutaraldehyde are popular fixatives for histology and pathology, the characteristics and fixation mechanisms are assumed to be quite different. Since formalin is composed of about 35% formaldehyde aqueous solution containing about 10% methanol to prevent the polymerization of formaldehyde and is usually diluted 10-fold as 10% formalin, its fixation mechanisms should be the same as those for 4% formaldehyde.
The mechanism of fixation using formaldehyde is thought to be as follows. Formaldehyde forms an adduction of hydroxymethyl/methylol (CH2OH) to functional groups of amino acids (such as lysine, arginine, and cysteine) (Figure 1a
Reaction of formaldehyde with proteins.
Although formaldehyde forms intra- and intermolecular crosslinks in proteins, the tertiary structures of the proteins are almost completely preserved [9, 11]. The methylene bridges between lysine and the phenyl residue of tyrosine are stable but most methylene bridges are unstable and reversible. Since basic residues of amino acids are modified with formaldehyde and the isoelectric point of proteins shifts to acidic, basic proteins should be precipitated at around the pH of the buffer (pH 7.2–7.4) used to dissolve the formaldehyde, based on the principle of isoelectric precipitation. Formaldehyde may first produce crosslinks among proteins in relatively stable core complexes, such as cell organelles, filament proteins in the cytoplasm and extracellular matrix, and chromatin, and then soluble proteins attach to these complexes to form a gel-like structure. Thus, these crosslinks interfere with the access of antibodies to antigens even if the epitopes do not have functional groups of amino acids that are directly modified by formaldehyde, as demonstrated in the model system by Sompran et al. using peptide epitopes coupled to glass slides [12].
FFPE specimens are assumed to be highly cross-linked, compared with formalin-fixed frozen sections. Since ethanol accelerates the imine formation of methylol groups and causes the rearrangement of the β-sheet of polypeptides and the exposure of hydrophobic amino acids, which are hidden in aqueous solutions, both intra- and intermolecular crosslinking should advance during dehydration and clearing with ethanol and xylene [13], and immersion in paraffin at around 60°C facilitates further crosslinking. On the other hand, some antigens in cell organelles might come in contact with antibodies more easily than those in frozen sections because membrane lipids are extracted and barriers are destroyed during dehydration.
Glutaraldehyde has been widely used as the standard primary fixative for electron microscopy specimens since introduced by Sabatini et al. in 1963 [14]. A mixture of glutaraldehyde and formaldehyde is also a popular fixative for cytology, enzyme cytochemistry, and immunoelectron microscopy. Glutaraldehyde (Figure 2a) has two aldehydes that can directly crosslink with the ε-amino residues of lysine and the N-terminus of polypeptides by forming a Schiff base. However, most investigators think that the rapid and extremely stable crosslinks formed by glutaraldehyde are based on the oligomeric form of glutaraldehyde. Kawahara et al. demonstrated that protein crosslinkage by forming the Schiff base and the aldol condensation of glutaraldehyde monomers occur almost in parallel and result in the formation of a linear glutaraldehyde oligomer with several Schiff base linkages branching off forming (-CH=CH-CH=N-R)n (Figure 2b), since glutaraldehyde solution showed no absorbance at 235 nm caused by α,β-unsaturated bonds in the absence of amines [15]. The resulting resonance structures are extremely stable to both heat and acid treatments [16].
Reaction of glutaraldehyde and osmium tetroxide with proteins and effect of heating. F-protein, fragmented protein by osmium tetroxide post-fixation.
Since the cross-linked proteins rapidly form harder gel-like structures, compared with those created by formaldehyde, only thin layers of tissues can be fixed well using immersion fixation. Since aldehyde residues remain in the tissues fixed with glutaraldehyde, the aldehyde should be quenched using amides, such as glycine, ammonium chloride, and tris(hydroxymethyl) aminomethane, or reduced to alcohols using sodium borohydride prior to immunostaining.
Since osmium tetroxide binds to the unsaturated bonds of fatty acids and fixes membrane lipids, providing contrast by scattering electron beams, it is used as the post-fixing reagent after glutaraldehyde fixation in electron microscopy. Osmium tetroxide should also bind to the carbon-carbon double bonds formed by glutaraldehyde fixation (Figure 2c). However, since osmium tetroxide cleaves polypeptides in tryptophan residues and oxidizes methionine to methionine sulfone and cysteine to cysteic acid [17], osmium tetroxide significantly inhibits immunoreactions.
After the first report for HIAR by Shi et al. [1], investigators have tried to select the most suitable heating conditions (heating devices, temperatures, kinds of solutions, solution pH, and additives). However, the total amount of applied heat energy is now recognized as being more important than the type of heating devices. In this section, the effects of pH and the ionic strength of retrieval solutions for HIAR will be reviewed, and the mechanisms of HIAR will be described.
When purified proteins are treated with formaldehyde and analyzed using SDS-PAGE (polyacrylamide gel electrophoresis), protein oligomers formed by intermolecular crosslinks were recognized. Monomer and oligomers treated with formaldehyde showed smaller apparent molecular weight compared with those of unmodified native proteins, since intramolecular crosslinks prevented the complete unfolding of proteins in the SDS solution [2, 18, 19]. The cleaving efficiency of the crosslinks was almost the same when the formaldehyde-treated proteins were heated for 5 min at 100°C in 10 mM Tris-HCl at pH 3.0, pH 6.0, pH 7.5, or pH 9.0 while analyzed with SDS-PAGE. When the proteins were drastically heated by autoclaving for 10 min at 120°C at a pH close to their respective isoelectric points, the proteins tended to produce insoluble protein precipitates [2]. However, many investigators have demonstrated that the efficiency of HIAR for immunohistochemistry is highly dependent on the pH of the retrieval solution.
Shi et al. systematically studied the effects of the pH of antigen retrieval solution on HIAR [20]. They classified the pH-influenced HIAR immunostaining patterns as follows: type A, in which staining was almost the same at any pH, with a slight decrease in intensity between pH 3.0 and pH 6.0; type B, in which a dramatic increase in immunostaining was observed at acidic and basic pH; and type C, in which the immunostaining intensity increased at basic pH. We re-examined the pH dependency of HIAR using 17 different antibodies and observed two immunostaining patterns for the pH dependency of HIAR [3]. The majority of the antibodies produced the first immunostaining pattern; that is, they yielded a positive immunoreaction when heated in buffers that had either an acidic pH or a basic pH. This HIAR pattern may correspond to the type-B pattern of the classification by Shi et al. If highly diluted antibodies had been used in the immunohistochemical studies, the type-A pattern described by Shi et al. might have become nearly equivalent to the type
Yamashita and Okada demonstrated that the intensities of the immunoreactions obtained by heating in a buffer are reversibly altered by successive heating in another buffer with a different pH [2]. For example, when the first heating in a buffer (pH 6.0) yielded a weak immunostaining in FFEP sections, a second heating at pH 9.0 significantly increased the immunostaining; however, the third heating in the acidic buffer weakened the immunostaining. These results indicate that the degradation or extraction of antigens is not a major factor in the pH dependency of HIAR and that the pH of the solution is a critical factor for the exposure of tissue epitopes in HIAR.
We studied the effects of ionic strength on HIAR using 10 antibodies. Three buffer systems with different pH values were examined. When FFPE specimens were autoclaved for 10 min at 120°C in 20 mM Tris-HCl buffer (pH 9.0), 50 mM citraconic anhydride aqueous solution (pH 7.4), or 10 mM citrate buffer (pH 6.0) containing 0, 50, 100, or 200 mM NaCl, all the antibodies showed the strongest immunostaining while the sections were autoclaved in the NaCl-free solutions. The staining intensity decreased as the NaCl concentration increased in all antibodies examined [24]. These results demonstrated that the ionic strength of the solution is a critical factor for HIAR and that a high concentration of salt inhibits the exposure of epitopes.
The results described above demonstrate that the fundamental mechanism of HIAR is based on the cleavage of protein-protein crosslinks and the exposure of epitopes. Heating destroys the gel-like structure formed by formaldehyde-fixation and partially extracts the macromolecules, enabling the antibodies to penetrate tissues easily; this process is similar to the effects of enzyme digestion. Western blot analyses have demonstrated that soluble, nuclear, and membrane proteins are extracted from FFPE specimens after heating but not from those without heat treatment [2]. Recent proteomics studies using a mass spectrum technique have also revealed that heating facilitates protein extraction from archived FFPE specimens [6]. Furthermore, heat-induced cleaving of the shortest crosslinks induced by formaldehyde can be applied to chromatin immunoprecipitation assays and to the crosslinks of adjacent proteins to investigate temporal interactions [8, 25, 26].
The second mechanism is assumed to be as follows based on the pH-dependent and ionic strength-dependent phenomena described above [3, 4]. When the methylene bridges are cleaved by heating, the higher order structure of the protein is destroyed and the polypeptide chains are extended, exposing both hydrophobic and hydrophilic regions and epitopes. The polypeptide chains then rapidly refold during the cooling process. In tissues, many kinds of proteins with different isoelectric points and molecular weights are tightly packed, and neighboring polypeptides can come in contact with each other. Therefore, epitopes should be concealed during the refolding of the proteins at around a neutral pH because a strong hydrophobic attractive force would randomly entangle the neighboring polypeptides: an electrostatic force may act locally as either an attractive or a repulsive force. At basic or acidic pH values, however, the majority of the extended polypeptides would be charged negatively or positively, and the electrostatic repulsive force would act to prevent random aggregation and entanglement with neighboring polypeptides caused by the hydrophobic force, thereby maintaining a suitable extend conformation for antigen-antibody interactions. When salt is added to the retrieval solutions, the electrostatic force between neighboring polypeptides is canceled, and the hydrophobic attractive force may cause the antigen proteins and neighboring proteins to aggregate, masking the epitopes. Namimatsu et al. reported that heating in citraconic anhydride solution at a neutral pH was useful as a universal antigen retrieval method [27]. Since citraconic anhydride binds to the ε-amino groups of lysine residues re-exposed after heating and places numerous negative charges on proteins, an electric repulsive force may help to keep polypeptides in an unfolded state.
On the other hand, strong heating at around the isoelectric points of proteins induces their coagulation [2], and increasing the ionic strength also promotes isoelectric precipitation. Therefore, many proteins with neutral isoelectric points can be precipitated in a solution with a neutral pH. The finding that an acidic buffer is effective for some basic antigens probably indicates that these antigens are precipitated by heating in basic buffers [23, 28]. Since heating may destroy the protein conformation, most conformational epitopes associated with noncovalent forces should lose their reactivity to antibodies. On the other hand, HIAR may be effective for conformational epitopes that have been stabilized by disulfide bonds.
Basic or acidic solutions are effective for HIAR as described above, whereas citrate buffer (pH 6.0) is frequently used in pathological studies. Citrate buffer may be suitable for examining detailed nuclear structures, since heating in basic solutions cleaves and extracts RNAs and reduces the nuclear stainability with hematoxylin. In practice, at least three antigen retrieval solutions at pH 3.0, pH 6.0, and pH 9.0 should be examined when studying the localization of unknown antigens for the first time.
Fresh frozen sections are widely used for immunohistochemical studies, because (1) they preserve antigenicity well, (2) they provide reproducible results because the fixation time is precisely controlled and the fixation is uniform throughout the sections, and (3) they allow antigen localization within a short time for pathological diagnosis. We introduced new fixative, 10% formalin containing 25 mM CaCl2 in 0.1 M HEPES-NaOH buffer (pH 7.4) that is more appropriate for the fixation of fresh frozen sections compared with buffered 10% formalin, because it has a stronger fixation ability and can crosslink proteins more rapidly and stabilize membranes, the extraction of antigens during fixation is minimized, and an excellent tissue structure is maintained after HIAR [29]. After heating in 20 mM Tris-HCl (pH 9.0) for 30 min or in 20 mM citrate buffer (pH 3.0), antigens in the nuclei, other cell organelles, cytoplasm, membranes, and extracellular matrix were clearly visible, even if they showed no immunoreactions without heating.
Few antigens that reduced disulfide bonds react with antibodies when they are analyzed using Western blotting (Figure 3A), whereas they reveal negative immunostaining even if the tissues were fixed with formaldehyde or other physical fixatives followed by heating (Figure 3B and D). In such cases, the epitopes should be located in a highly folded portion of the antigen protein that may be stabilized with disulfide bonds to form stable secondary and tertiary structures. The reduction of disulfide bonds using dithiothreitol or 2-mercaptoethanol prior to heat treatment yields strong immunoreactions (Figure 3C and E) [28, 30]. In addition, when epitopes are covered by neighboring heat-resistant polypeptides, the accessibility of antibodies to the epitopes is also inhibited. When immunostaining is performed using old FFEP or using sections on a slide glass stored for a long time, the samples should be oxidized to produce many disulfide bonds in the tissues. The reduction and cleavage of disulfide bonds may be effective for such specimens (Figure 3F–I).
Antigen retrieval for highly masked epitopes with disulfide bonds in paraffin and frozen sections. A. VEGFR1 and Tie 2 expressions in the uteri of 10-day-old mice were demonstrated using Western blotting. VEGFR1 and Tie 2 proteins were analyzed after treatment with 2-mercaptoethanol (lines 2 and 4) or without the treatment (lines 1 and 3), respectively. Membrane bound VEGFR1 (150 kD) shows no reaction to the antibody without reduction of disulfide bonds, whereas soluble VEGFR1 (100 kD) binds to the antibody independence of reduction (lines 1 and 2). Tie 2 antibody shows strong immunoreaction to Tie 2 protein-reduced disulfide bonds (lines 3 and 4). B–E. Immunostaining of VEGFR1 (B and C) and Tie 2 (D and E) in the frozen sections of 10-day-old mouse uteri. Fresh frozen sections were fixed with 10% formalin containing 25 mM CaCl2 in 0.1 M HEPES buffer (pH 7.4) for 5 min, treated with 0.1 M dithiothreitol (DTT)/20 mM Tris-HCl (pH 9.0) for 1 h and then with 0.1 M iodoacetamide/0.1 M Tris-HCl (pH 9.0) for 15 min (C and E); B and D, sections without reduction. The sections were then heated in 20 mM Tris-HCl (pH 9.0) for 30 min at 95°C and immunostained with the antibodies. The stroma shows diffuse VEGFR1 immunostaining which may be based on the soluble VEGFR1 in non-reduced section (B), while vasculatures (arrows) and basal membrane of epithelial cells exhibit positive VEGFR1-immunostaining which may be corresponded to the membrane-bound VEGFR1 (C). Clear Tie 2-immunostaining is recognized in the endothelial cells of vasculatures (arrows) in the reduced section (E) but not in the non-reduced section (D). F–I. Immunohistochemistry in paraffin sections. Clathrin was detected in the mouse pancreas after reduction with DTT (G) or without reduction (F) followed by heating in 20 mM Tris-HCl (pH 9.0). Tom 20 was immunostained in the mouse ovary after reduction with DTT (I) or without reduction (H) followed by heating. Bar = 50 μm.
Immunoelectron microscopy is a powerful technique for observing the localization of antigens in cell organelles and for studying the relationship between antigens and other macromolecules. Three main immunoelectron microscopy methods have been used to localize antigens: the pre-embedding method, the post-embedding method, and cryoultramicrotomy. Although fixation is one of the most important aspects of sample preparation for all three methods, the choices of fixatives and tissue processing procedures are limited unlike light microscopy, because of the need to satisfy compatible requirements, such as conservation of fine structure, immobilization of antigen minimizing the diffusion, and conservation of immunoreaction. In general, fixatives that allow good morphological findings and precise antigen localization through the rapid and tight crosslinking of macromolecules also severely inhibit antigen-antibody interactions.
Fixatives containing formaldehyde as the main crosslinking regent are popular for immunoelectron microscopy using the pre-embedding method, since antibody penetration into the cells is an important factor for this method; formaldehyde solution, PLP (periodate-lysine-paraformaldehyde) [31], and a mixture of formaldehyde and a low concentration (0.05–0.5%) of glutaraldehyde. For the post-embedding method, a mixture of formaldehyde and a low concentration of glutaraldehyde is frequently used to preserve the fine structure and membrane structures, since dehydration and resin embedding are performed without osmium tetroxide post-fixation. A short period of perfusion fixation with glutaraldehyde is also applied for the post-embedding method. The suitable fixatives, fixing periods, and temperatures of fixatives have been determined by trial and error for each antigen independent of the staining methods.
In this section, a standardized fixation method that yields positive immunoreactions for the pre-embedding and the post-embedding methods after HIAR will be described and discussed how HIAR is also effective for some routinely processed materials for the electron microscopy that are fixed with glutaraldehyde and osmium tetroxide and embedded in epoxy resins.
We introduced a standardized fixative that can yield positive immunoreactions for many antigens in electron microscopy after HIAR [32]. Tissues were fixed with 4% formaldehyde containing 2.5 mM CaCl2 and 1.25 mM MgCl2 in 0.1 M HEPES-NaOH buffer (pH 7.4) for 2 h at room temperature and then with the same fixative composition in 0.1 M HEPES buffer-NaOH buffer (pH 8.5) overnight at room temperature. The vehicle osmolarity of the fixatives was adjusted to 300–330 mOsm by adding sucrose or glucose. Formaldehyde containing CaCl2 and MgCl2 was shown to be the best fixative, producing a rapid and complete fixation that minimizes diffusion artifacts (the divalent cations are well known to act as stabilizers of membrane structures). In addition, tissues were fixed using two steps: first at pH 7.4 and then at pH 8.5. This method preserves the cellular fine structures because the crosslinking reaction produced by formaldehyde progresses rapidly at a basic pH. Fixation was then performed at room temperature to enable a faster reaction than that possible at 4°C.
Although the pre-embedding method is the most popular and the simplest method for immunoelectron microscopy, HIAR has only been applied for the detection of a few antigens. Frozen sections or vibratome sections from specimens fixed with formaldehyde or a mixture of formaldehyde and glutaraldehyde were heated in various solutions such as citrate buffer (pH 6.0), Tris-HCl buffer (pH 9.0 or pH 10.0), or citraconic anhydride solution (pH 7.4) for different periods for each antigen [33, 34, 35, 36]. Yamashita reported that 4% formaldehyde containing 25 mM CaCl2 in 0.1 M cacodylate buffer (pH 7.4) was a suitable fixative for the pre-embedding method by applying HIAR for several antigens [4].
We applied the pre-embedding method to tissues fixed using the standardized fixative described above. Frozen sections (about 15 μm) were mounted on a slide glass and then heated in 20 mM Tris-HCl (pH 9.0) containing 10% sucrose for 2–4 h at 70°C. Immunostaining was performed using (horseradish peroxidase) HRP-labeled antibodies and antigen localization was visualized with 3,3′diaminobenzidine (DAB). Most of the antigens that were examined showed negative immunoreactions without heat treatment, but they produced strong immunoreaction after heating (Figure 4). Since endogenous immunoglobulins are inactivated after heat treatment (Figure 4A and B), the immunoreactions can be clearly detected even in the mouse tissues using mouse monoclonal antibodies. Tris-HCl buffer (pH 9.0) is effective for most antigens but citrate buffer (pH 6.0 or pH 3.0) yields strong reaction for a few antigens with basic isoelectric points, such as vascular endothelial cell growth factor (VEGF). Therefore, the selection of suitable solutions for each antigen should be examined using FFEP sections or frozen sections on light microscopy.
HIAR in frozen sections form specimens fixed with the standardized fixative. Mouse tissues were fixed with the standardized fixative and immersed in 10, 15, and 20% sucrose and then frozen. Frozen sections (15 μm) were immunostained with anti-E-cadherin monoclonal antibody in the small intestine (A and B), anti-β-catenin monoclonal antibody in the pancreas (C and D), and anti-claudin 5 polyclonal antibody in the kidney (E and F) after heating in 10 mM Tris-HCl buffer (pH 9.0) containing 10% sucrose for 3 h at 70°C (B, D, and F) or without heating (A, C, and E). Endogenous mouse immunoglobulins are seen in the lamina propria mucosa (arrows) and plasma cells (outline arrows), but E-cadherin immunoreactions is not detected without heating (A). However, the endogenous immunoglobulin immunostaining is negative and E-cadherin is clearly localized along the lateral membrane of intestinal epithelium after heating; the junctional complexes show strong immunoreactions (B). β-Catenin immunoreaction is present along the lateral membrane of pancreatic acinar cells (D) but not in the section without heating (C). After heating, claudin-5 is localized in the glomeruli and distal tubules in the kidney (F). Bar = 50 μm.
The positively immunostained sections were then post-fixed with 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4) for 30 min, dehydrated with ethanol, and then embedded in epoxy resin. All antigens detected in frozen sections on light microscopy were localized using the pre-embedding method preserving fine structures (Figure 5).
HIAR for immunoelectron microscopy with the pre-embedding method. Frozen sections immunostained with anti-E-cadherin antibody (A) and anti-claudin-5 antibody (B) demonstrated in
The post-embedding method provides more reproducible and reliable immunostaining results than the pre-embedding method, which produces a limited and heterogeneous penetration of antibodies into the tissues, since immunoreactions occur on the surfaces of ultrathin sections. Furthermore, immunoreactions on ultrathin sections permit the counting of immunogold particles, enabling semi-quantitative analyses, and the simultaneous staining of multiple antigens. Although the post-embedding method has these advantages, it has only been used for a limited number of antigens because antigenicity is frequently lost during the dehydration and embedding procedures. Specimens embedded in acrylic resins without osmication show a disrupted membrane structure and poorly contrasted cell organelles.
Several investigators have applied HIAR to the post-embedding method and have reported its usefulness [37, 38, 39]. Tissues were fixed with a mixture of formaldehyde and glutaraldehyde or formaldehyde alone and embedded in acryl resins; then, ultrathin sections were heated in various solutions. We attempted to establish a standardized method for immunoelectron microscopy that would satisfy the following requirements: (1) the preservation of fine cell structures with good image contrast in tissues embedded in acryl resins without OsO4 post-fixation, (2) the application of HIAR to obtain a high labeling density, and (3) a simple and reproducible method that does not require special equipment [32, 39].
Tissues were fixed with the standardized fixative and dehydrated with dimethylformamide (DMF) on ice and embedded in the LR-White resin, since DMF may reduce abrupt osmotic pressure changes in the tissues and the extraction of membrane lipids. The resin was polymerized for 24 h at 55°C. Ultrathin sections mounted on a nickel grid were heated in 0.5 M Tris-HCl buffer (pH 9.0) for 1–2 h at 95°C. After immunogold labeling, the sections were treated with 2% glutaraldehyde containing 0.05% tannic acid in 0.1 M phosphate buffer (pH 5.5) for 5 min and with 1% OsO4/0.1 M phosphate buffer (pH 7.4) for 5 min and then double stained with uranyl acetate and lead citrate. This method yielded strong and reproducible immunoreactions for many soluble, membrane bound, and filamentous proteins (Figure 6), [32, 39]. Furthermore, tannic acid treatment followed by osmium tetroxide treatment produced good contrasted images. The cellular membranes produced a positive image and the cell organelles, such as mitochondria, the Golgi complexes, secretory granules, and lysosomes, were well contrasted. Nucleic acids (chromatins, nucleoli, and ribosomes), intracellular filaments (actin filaments, 10 nm filaments, and microtubules), and collagen fibers were well visualized.
HIAR for immunoelectron microscopy with the post-embedding method. Mouse kidney was fixed with the standardized fixative and then embedded in LR-White resin. Ultrathin sections were heated in 0.5 M Tris-HCl (pH 9.0) for 1 h at 95°C and treated with anti-β-actin/TBS (A), Tom 20/10 mM Tris-HCl (pH 7.4) containing 50 mM NaCl (B), and anti-mortalin (mitochondrial 70 kD heat shock protein)/TBS (C) and then treated with colloidal gold-labeled secondary antibodies/TBS. A. Strong β-actin immunoreactions are seen in the foot processes of podocyte (P) (outline arrows) and in the cytoplasm of mesangial cell (M). Lymphocyte (L) shows potty reactions beneath the cell membrane (arrows). B. Tom 20 is localized along the mitochondria but not recognized in the lysosome (outline arrow). C. Mitochondria show mortalin immunoreaction, whereas lysosomes (outline arrows), apical canaliculi (arrows), and nucleus are negative for staining. Bars = 500 nm.
Archives of materials embedded in epoxy resins are collected in many histology and pathology laboratories and in hospitals for morphological analyses, and these archives are expected to provide valuable data if they can be used for immunohistochemical studies. However, conventionally processed specimens for transmission electron microscopy have been regarded as unsuitable for immunoelectron microscopy using post-embedding methods for the following reasons: (1) Glutaraldehyde significantly suppresses antigen-antibody interactions and HIAR is ineffective for most antigens. (2) Osmium tetroxide severely inhibits immunoreactions by cleaving polypeptides and oxidizing methionine and cysteine [17]. (3) Epoxy resins produce tight three-dimensional crosslinks that suppress antigen-antibody interactions.
A limited number of antigens can be successfully detected on sections from conventionally processed materials. One method involves the oxidation and removal of osmium by treating ultrathin sections with sodium metaperiodate aqueous solution [40]. Another method involves the partial removal of epoxy resins by treating the sections with hydrogen peroxide or sodium/potassium ethoxide [41, 42]. The combined use of partial deresination and heat treatment has also been examined. Borson and Skjorten polymerized the epoxy resin to reduce copolymerization between the proteins and the resin and to make a porous polymer for applying HIAR to glutaraldehyde-fixed and epon-embedded materials [43]. We have also demonstrated that HIAR is effective for post-embedding immunoelectron microscopy using conventionally processed epon blocks, contrary to presumptions that antigen detection would be a special case in these specimens [44].
The effectiveness of HIAR in the post-embedding method using conventionally processed epon-embedded specimens was systematically examined for 18 antibodies. Frozen sections fixed with 2% glutaraldehyde for 30 min at room temperature or with 2% glutaraldehyde for 30 min followed by 1% osmium tetroxide for 30 min at room temperature were dehydrated with ethanol and then rehydrated to elucidate the validity of HIAR for avoiding the effects of epoxy resin embedment. In another experiment, frozen sections fixed with glutaraldehyde and osmium tetroxide were treated with sodium metaperiodate, since this reagent has been reported to be effective for osmicated materials, as described above.
After autoclaving in Tris-HCl buffer (pH 9.0), 7 of the 18 antibodies exhibited a strong immunoreaction in frozen sections fixed with glutaraldehyde and osmium tetroxide, whereas heating revealed almost no effect on the sections fixed with glutaraldehyde alone (Table 1; Figure 7). Treatment with sodium metaperiodate was ineffective for the antigen retrieval of all the antibodies (Table 1). The mechanisms of HIAR in the frozen sections fixed with glutaraldehyde and osmium tetroxide were assumed to be as follows [44]. Osmium tetroxide binds to ethylene bonds formed by glutaraldehyde fixation, i.e., -CH=CH-CH=N-R (Figure 2c). Heat treatment removes the osmium tetroxide additives and forms 1, 2-diols (Figure 2d), since the black color fades in frozen sections fixed with glutaraldehyde and osmium tetroxide after autoclaving. The cleaving of the double bonds should extend the antigen polypeptides and increase the flexibility of the polypeptides. However, the morphology in the frozen tissues fixed with glutaraldehyde and osmium tetroxide was more disrupted after autoclaving, compared with those fixed with glutaraldehyde alone or 4% formaldehyde containing 25 mM CaCl2. The reason may be as follows. Osmium tetroxide treatment induces polypeptide fragmentation (Figure 2c and d), and heating extracts the fragments after crosslink cleavage by glutaraldehyde.
GA-EtOH | GA-OsO4-EtOH | ||||
---|---|---|---|---|---|
Non | Autoclave | Non | NaIO4 | Autoclave | |
PCNA | 0 | 1 | 0 | 0 | 2 |
Clathrin | 0 | 0–1 | 0 | 0 | 3 |
GFAP | 0 | 0–1 | 0 | 0 | 3 |
Occludin | 0 | 0–1 | 0 | 0 | 3 |
Tom 20 | 0 | 0–1 | 0 | 0 | 3 |
Claudin-5 | 0 | 0 | 0 | 0 | 2 |
α-Amylase | 0–1 | 1 | 0 | 0–1 | 2 |
NRP1 | 0 | 0 | 0 | 1 | |
β-Catenin | 0–1 | 0 | 0 | 0 | |
E-Cadherin | 0 | 0–1 | 0 | 0 | |
Desmin | 0 | 0–1 | 0 | 0 | |
Caveolin | 0 | 0 | 0 | 0 | |
VEGFR2 | 0–1 | 0 | 0 | 0 | |
ERα | 0 | 1–2 | 0 | 0–1 | |
AnR | 0 | 0 | 0 | 0 | |
β-Actin | 1 | 1 | 0 | 0 | |
γ-GTP | 1 | 1 | 0 | 0 |
HIAR in frozen sections prepared from specimens fixed with glutaraldehyde or glutaraldehyde and osmium tetroxide.
Fresh frozen sections were fixed with 2% glutaraldehyde/0.1 M phosphate buffer (pH 7.4) for 30 min (GA-EtOH) or fixed with 2% glutaraldehyde/0.1 M phosphate buffer (pH 7.4) for 30 min and further fixed with 1% osmium tetroxide/0.1 M phosphate buffer (pH 7.4) for 30 min (GA-OsO4-EtOH). Some sections fixed with glutaraldehyde and osmium tetroxide were further treated with 1% sodium metaperiodate aqueous solution for 5 min (NaIO4): all sections were hydrated with ethanol and then rehydrated after fixation. The fixed sections were immunostained after autoclaving in 20 mM Tris-HCl (pH 9.0) at 120°C for 10 min (autoclave) or without heat treatment (Non). Immunostaining was scored as followed: 3, strong; 2, moderate; 1, weak; 0–1, faint; and 0, negative.
HIAR in frozen sections fixed with glutaraldehyde and osmium tetroxide. Fresh frozen sections (6 μm) from mouse tissues were fixed with 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 min at room temperature (A, C, and E) and successively with 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4) for 30 min at room temperature (B, D, and F). The sections were autoclaved in 20 mM Tris-HCl (pH 9.0) for 10 min at 120°C. They were then immunostained with anti-α-amylase antibody in the pancreas (A and B), anti-claudin-5 antibody in the kidney (C and D), and anti-clathrin antibody in the kidney (E and F). Although negative or weak immunostaining is seen in the sections fixed with glutaraldehyde after autoclave (A, C, and E), strong α-amylase immunoreactions are recognized in the apical cytoplasm of pancreatic acinar cells (B) and clear claudin-5 and clathrin immunoreactions are observed in the glomeruli (D) and in the apical cytoplasm of proximal tubular cells in the kidney (F), respectively. Bar = 50 μm.
Partial deresination with sodium ethoxide was required for light microscopy using semi-thin epon sections. After autoclaving in 100 m Tris-HCl (pH 9.0) for 10 min, six of the seven antibodies that showed positive immunoreactions in the frozen sections exhibited clear localizations in the semi-thin sections. α-Amylase (Figure 8A and B), clathrin (Figure 8C), and claudin-5 (Figure 8D), which all showed positive immunoreactions in the semi-thin sections, were localized on ultrathin sections using colloidal gold-labeled antibodies. For HIAR, ultrathin sections were heated in 500 mM Tris-HCl buffer (pH 9.0) for 1–3 h at 95°C. Although heat treatment was essential for the detection of antigens on ultrathin sections, heat treatment reduced the electron density of ribosomes, chromatins, intracellular membranes, and secretory granules in the exocrine pancreas. The partial removal of epoxy resins with sodium ethoxide followed by autoclaving revealed the disruption of the fine structure and no reproducible immunolabeling.
HIAR for the specimens fixed with glutaraldehyde and osmium tetroxide, embedded in epoxy resin. Mouse tissues were fixed with 2% glutaraldehyde/0.1 M phosphate buffer (pH 7.4) for 3 h at 4°C and post-fixed with 1% osmium tetroxide/0.1 M phosphate buffer (pH 7.4) for 1 h and then embedded in the epoxy resin. Ultrathin sections were heated in 0.5 M Tris-HCl (pH 9.0) for 2 h at 95°C. α-Amylase is localized in the Golgi apparatus (G), condensing vacuole and secretory granules in the exocrine pancreas after heat treatment (B), whereas no reaction is seen in the sections without heating (A). Immunoreaction for clathrin is recognized in the apical canaliculi of renal proximal tubular cell (C). Claudin-5 is localized along the membrane of podocyte foot processes in the glomerulus (D). Bar = 500 nm.
These results indicated that archived epon-embedded specimens could be a useful resource for immunohistochemical studies at both the light and electron microscopy levels, since they provide excellent morphology and detailed antigen localization compared with paraffin-embedded materials.
The relationship between epitopes and paratopes of antibodies is thought to be similar to that between keys and keyholes. However, since these structures change their conformations to form a final specific and tight binding after antigen-antibody association, conservation of the flexibility of their polypeptide chains should be important. Although hydrogen bonds, hydrophobic forces, electrostatic forces, and van der Waals forces all participate in the final tight binding, electrostatic forces are important for the initial contact and association of antigen and antibody molecules (i.e., the net charges of each molecule and the neighboring charges of antigens). Buffer type, ionic strength, pH, and the presence of detergents in solutions are likely to exert strong influences on the antigen-antibody reaction. Although many kinds of diluents are commercially available for immunohistochemistry and Western blotting and yield good results with low background staining and a high sensitivity for some antigens, systematic studies of antibody diluents for immunohistochemistry have not been performed. In this section, the effects of dilution solutions for primary antibodies on immunostaining for light and electron microscopy are described.
Fifteen monoclonal antibodies were diluted in 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl (PBS), 10 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl (TBS), or 10 mM FEPES-NaOH buffer (pH 7.4) containing 150 mM NaCl (HBS); 1% bovine serum albumin (BSA) (final concentration) was added to each solution. Paraffin sections from mouse tissues fixed with formaldehyde were immunostained after autoclaving in 20 mM Tris-HCl buffer (pH 9.0) for 10 min. The sections were treated with the primary antibodies diluted with the solutions overnight at 4°C and successively with Envision HRR (Dakocytomation) for 1 h at room temperature. As shown in Table 2, all the antibodies diluted with TBS showed stronger immunoreactions than those diluted with PBS or HBS [45]. Although the reasons are unclear, the binding of phosphate ion (a larger ion) to positively charged regions of epitopes and paratopes may reduce the flexibility of peptide chains.
Antibodies | Clones and subclasses | Dilution buffer type | mM NaCl/10 mM TB | ||||
---|---|---|---|---|---|---|---|
PBS | TBS | HBS | 50 | 150 | 300 | ||
PCNA | PC10; IgG2a | 1 | 2 | 2 | 1 | 2 | 2–3 |
ERα | D12; IgG2a | 3 | 2 | 2 | 2 | 2 | 2 |
ERα | ID5; IgG1 | 1 | 1–2 | 1 | 2 | 1-2 | 1 |
S-100 | M2A10; IgG1 | 1 | 2 | 1–2 | 3 | 2 | 1 |
Mortalin | JG1; IgG3 | 3 | 3 | 3 | 3 | 3 | 3 |
HSP 70 | sc-27; IgG2a | 1 | 2 | 2 | 3 | 2 | 1–2 |
α-Synuclein | 42; IgG1 | 2 | 2 | 2 | 2 | 2 | 2 |
GFAP | 6F2; IgG1 | 2 | 2 | 1 | 1 | 2 | 2 |
Desmin | D33; IgG1 | 1 | 2 | 1 | 1 | 2 | 2 |
β-Actin | AC-74; IgG2a | 2 | 2 | 2 | 1 | 2 | 1–2 |
Clathrin | X22; IgG1 | 1 | 2 | 2–3 | 3 | 2 | 1 |
E-Cadherin | 36B5; Ig1 | 1 | 2 | 2 | 3 | 2 | 1 |
β-Catenin | sc-763; IgG1 | 1 | 2 | 1 | 3 | 2 | 1 |
γ-GTP | 5B9; IgG1 | 1 | 2 | 2 | 3 | 2 | 1 |
CASGM | 170-5; IgG1 | 2 | 2 | 2 | 3 | 2 | 1 |
Effects of diluents for monoclonal antibodies on immunohistochemistry.
FFPE sections (6 μm) of mouse tissues were autoclaved in 20 mM Tris-HCl (pH 9.0) for 10 min and then immunostained with monoclonal antibodies. Antibodies were diluted with 150 mM NaCl/10 mM phosphate buffer (pH 7.4) (PBS), 150 mM NaCl/10 mM Tris-HCl (pH 7.4) (TBS), or 150 mM NaCl/10 mM HEPES buffer (pH 7.4) (HBS). The antibodies were also diluted with 10 mM Tris-HCl (pH 7.4) containing 50 mM, 150 mM, or 300 mM NaCl. Immunostaining was scored as followed: 3, strong; 2, moderate; and 1, weak.
Fifteen monoclonal antibodies were diluted in 1% BSA/10 mM Tris-HCl buffer (pH 7.4) containing 50 mM NaCl, 150 mM NaCl, or 300 mM NaCl. After autoclaving, the paraffin sections were treated with the primary antibodies overnight and then with Envision HRR for 1 h at room temperature. The results are shown in Table 2. Most of the antibodies showed strong immunostaining when they were diluted with a buffer containing 50 mM NaCl [45]. However, monoclonal antibodies to proliferating cell nuclear antigen (PCNA) showed the strongest immunostaining when diluted with a buffer containing 300 mM NaCl, and monoclonal antibodies to glial fibrillary acidic protein (GFAP) and β-actin diluted with 150 mM yielded the strongest immunostaining. Polyclonal antibodies to nuclear transcription factors such as estrogen receptor (ER)α, androgen receptor (AnR), glucocorticoid receptor, and p300 yielded stronger immunostaining when diluted with a buffer containing 150 or 300 mM NaCl than that using a buffer containing 50 mM NaCl (not shown). These results suggest that the net charges of antibodies and antigens influence the contact of these proteins, and the net neighboring charges of antigens also affect the interactions. Nuclear antigens are associated with highly acidic nucleic acid, and β-actin and GFAP bundles are composed of β-actin and GFAP proteins with acidic isoelectric points. Dilution solution with a high ionic strength may reduce the net charges around the antigens and antigen molecules, allowing antibodies to come in contact with their respective antigens.
In immunohistochemistry using HRP-labeled antibodies, the staining intensity can be increased using highly sensitive reaction solutions and a longer enzyme reaction time. However, the intensification of immunoreactions is difficult in the post-embedding method and ultracryotomy using colloidal gold-labeled secondary antibody. Instead, the selection of diluents for the antibodies may be more important for obtaining a high labeling density with a low background staining compared with light microscopy, whereas almost no studies have been performed for immunoelectron microscopy.
We examined the effect of diluents of several antibodies for the post-embedding method using specimens fixed with the standardized fixative and embedded in LR-White resin [39, 41]. Glutaraldehyde and osmium tetroxide fixed and epon-embedded specimens were also used for a few antibodies. After HIAR, ultrathin sections were immunostained using antibodies diluted in the following solutions: PBS, TBS, 10 mM Tris-HCl (pH 7.4) containing 50 mM NaCl, Can Get Signal A (Toyobo Co.), and Can Get Signal B (Toyobo Co.); 1% BSA (final concentration) was then added to the diluents. In general, diluents that produced strong immunoreactions on light microscopy also produced a high labeling density of colloidal gold-labeled antibody. Anti-claudin-5 polyclonal antibody showed the strongest immunoreaction when it was diluted with Can Get Signal A for both LR-White-embedded materials and epon-embedded materials (Figure 8D). Can Get Signal A also showed the strongest immunoreactions when used as the diluent for E-cadherin monoclonal antibody. Monoclonal antibodies to β-catenin, β-actin, and clathrin showed a high labeling density when diluted in TBS. Polyclonal antibody to Tom 20 diluted with 50 mM NaCl/10 mM Tris-HCl (Figure 6B) or Can Get Signal B showed strong immunostaining. TBS was a better diluent for colloidal gold-labeled secondary antibodies than PBS.
The main mechanisms of HIAR are cleavage of chemical crosslinks formed by formaldehyde and extend of polypeptides chains to expose epitopes. Highly masked epitopes in heat-stable proteins can be exposed with reduction of disulfide bond followed by heating. Heating also cleaves crosslinks formed by double fixation with glutaraldehyde and osmium tetroxide. The principle of HIAR is applicable for immunoelectron microscopy using the post-embedding and pre-embedding methods, in which tissues are fixed with a standardized fixative described in this text. The association of exposed epitopes and antibodies under a suitable solution are also important for each immunohistochemical reaction.
AnR | androgen receptor |
BSA | bovine serum albumin |
CAGSM | common antigen of secretory granule membrane |
DAB | 3,3′ diaminobenzidine |
ERα | estrogen receptor α |
FFPE | formalin-fixed and paraffin embedded |
GFAP | glial fibrillary acidic protein |
γ-GTP | γ-glutamyl transpeptidase |
HIAR | heat-induced antigen retrieval |
HRP | horseradish peroxidase |
HSP | heat shock protein |
NRP | neuropilin |
PBS | phosphate-buffered saline (10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl) |
PCNA | proliferating cell nuclear antigen |
Tom | translocase of outer mitochondrial protein |
VEGF | vascular endothelial cell growth factor |
VEGFR | VEGF receptor |
TBS | Tris-buffered saline (10 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl) |
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In cases, the ultrasound appearance is a cystic image with different content and the differential diagnosis is often difficult. Body—research methods: the organs affected by abdominal congenital anomalies involve the gastrointestinal tract (stomach, duodenum, small bowel or colon, and gall bladder), the kidney and urinary tract, the peritoneal cavity (ascites), suprarenal glands, and tumors of the reproductive system (especially the ovaries). In order to identify the affected structures, it is mandatory to know the normal aspect of the abdominal content at different gestational ages. The diagnosis may be very difficult, but its accuracy is important, considering the need of further counseling the couple. In minor conditions, without chromosomal anomalies or associations, the outcome is usually good, and there are even possibilities of in utero treatment. In severe conditions, with poor outcome, the couple can choose to terminate the pregnancy, after counseling is provided. Conclusion: abdominal congenital anomalies are common findings in ultrasound screenings for anomalies in all the trimesters of pregnancy and their recognition is important for subsequent management.",book:{id:"6307",slug:"congenital-anomalies-from-the-embryo-to-the-neonate",title:"Congenital Anomalies",fullTitle:"Congenital Anomalies - From the Embryo to the Neonate"},signatures:"Ples Liana and Anca Lesnic",authors:[{id:"212333",title:"Associate Prof.",name:"Liana",middleName:null,surname:"Ples",slug:"liana-ples",fullName:"Liana Ples"}]},{id:"64417",title:"Introductory Chapter: A Comprehensive Approach to the Process of Breastfeeding",slug:"introductory-chapter-a-comprehensive-approach-to-the-process-of-breastfeeding",totalDownloads:1267,totalCrossrefCites:0,totalDimensionsCites:0,abstract:null,book:{id:"6191",slug:"selected-topics-in-breastfeeding",title:"Selected Topics in Breastfeeding",fullTitle:"Selected Topics in Breastfeeding"},signatures:"René Mauricio Barría P",authors:[{id:"88861",title:"Dr.",name:"R. Mauricio",middleName:null,surname:"Barría",slug:"r.-mauricio-barria",fullName:"R. Mauricio Barría"}]},{id:"62854",title:"The Surgical Technique of Caesarean Section: What is Evidence Based?",slug:"the-surgical-technique-of-caesarean-section-what-is-evidence-based-",totalDownloads:2448,totalCrossrefCites:1,totalDimensionsCites:1,abstract:"Caesarean section is the most frequent obstetric operation which is associated with increased maternal morbidity and mortality. Although these risks are low, affected women may suffer from severe consequences and this may affect subsequent pregnancies and deliveries. A variety of surgical approaches have been described, however, on low evidence level. The objective of this chapter is therefore to systematically search the literature and analyse the available evidence including preoperative workup, prophylactic antibiotics, skin disinfection, preoperative bladder catheterization as well as details of the individual steps of the actual operation itself such as skin incision types, preparation of soft tissue and womb, removal of the placenta, cervical dilatation and stitching of the womb, peritoneum, rectus muscle, fascia, subcutaneous fat, and skin. We systematically searched for meta-analysis, systematic reviews, and big studies and evaluated the evidence for each individual step.",book:{id:"6707",slug:"caesarean-section",title:"Caesarean Section",fullTitle:"Caesarean Section"},signatures:"Jan-Simon Lanowski and Constantin S. von Kaisenberg",authors:[{id:"100660",title:"Prof.",name:"Constantin",middleName:"Sylvius",surname:"Von Kaisenberg",slug:"constantin-von-kaisenberg",fullName:"Constantin Von Kaisenberg"},{id:"240353",title:"Dr.",name:"Jan-Simon",middleName:null,surname:"Lanowski",slug:"jan-simon-lanowski",fullName:"Jan-Simon Lanowski"}]},{id:"18348",title:"Anaesthetic Considerations during Laparoscopic Surgery",slug:"anaesthetic-considerations-during-laparoscopic-surgery",totalDownloads:28882,totalCrossrefCites:1,totalDimensionsCites:5,abstract:null,book:{id:"916",slug:"advanced-gynecologic-endoscopy",title:"Advanced Gynecologic Endoscopy",fullTitle:"Advanced Gynecologic Endoscopy"},signatures:"Maria F. Martín-Cancho, Diego Celdrán, Juan R. Lima, Maria S. Carrasco-Jimenez, Francisco M. Sánchez-Margallo and Jesús Usón-Gargallo",authors:[{id:"14715",title:"Prof.",name:"Francisco M.",middleName:null,surname:"Sánchez-Margallo",slug:"francisco-m.-sanchez-margallo",fullName:"Francisco M. Sánchez-Margallo"},{id:"29449",title:"Dr.",name:"Maria Fernanda",middleName:null,surname:"Martín-Cancho",slug:"maria-fernanda-martin-cancho",fullName:"Maria Fernanda Martín-Cancho"},{id:"39772",title:"Dr.",name:"Juan R.",middleName:null,surname:"Lima",slug:"juan-r.-lima",fullName:"Juan R. Lima"},{id:"39773",title:"Mr.",name:"Diego",middleName:null,surname:"Celdran",slug:"diego-celdran",fullName:"Diego Celdran"},{id:"39774",title:"Prof.",name:"Jesus",middleName:null,surname:"Usón-Gargallo",slug:"jesus-uson-gargallo",fullName:"Jesus Usón-Gargallo"},{id:"62320",title:"Prof.",name:"Maria Sol",middleName:null,surname:"Carrasco-Jiménez",slug:"maria-sol-carrasco-jimenez",fullName:"Maria Sol Carrasco-Jiménez"}]},{id:"41721",title:"Artificial Insemination in Poultry",slug:"artificial-insemination-in-poultry",totalDownloads:9531,totalCrossrefCites:5,totalDimensionsCites:14,abstract:null,book:{id:"3206",slug:"success-in-artificial-insemination-quality-of-semen-and-diagnostics-employed",title:"Success in Artificial Insemination",fullTitle:"Success in Artificial Insemination - Quality of Semen and Diagnostics Employed"},signatures:"M.R. Bakst and J.S. Dymond",authors:[{id:"155683",title:"Dr.",name:"Murray R.",middleName:null,surname:"Bakst",slug:"murray-r.-bakst",fullName:"Murray R. Bakst"},{id:"167852",title:"Dr.",name:"Jessica",middleName:null,surname:"Dymond",slug:"jessica-dymond",fullName:"Jessica Dymond"}]}],onlineFirstChaptersFilter:{topicId:"189",limit:6,offset:0},onlineFirstChaptersCollection:[{id:"80860",title:"From Open to Minimally Invasive: The Sacrocolpopexy",slug:"from-open-to-minimally-invasive-the-sacrocolpopexy",totalDownloads:35,totalDimensionsCites:0,doi:"10.5772/intechopen.101308",abstract:"With an increased demand for pelvic organ prolapse surgeries as the population ages, mesh-related osteomyelitis will become more prevalent. This case series enriches the paucity of data on management options for delayed osteomyelitis related to pelvic organ prolapse mesh. A literature review revealed no case reports of delayed onset osteomyelitis presenting up to a decade after colpopexy mesh placement. We present three cases of delayed osteomyelitis, their presentation, diagnosis and management at a tertiary academic referral center. Patients presented between 1 and 10 years after mesh colpopexy. Three different mesh materials were utilized during the initial procedures: Restorelle Y, Gynamesh and Gore-Tex mesh. The first case demonstrates failed expectant management with eventual surgical intervention on a medically compromised patient. The two subsequent cases describe elective complete mesh resection after several prior failed mesh revision attempts. This short case series and literature review illustrates that mesh-related osteomyelitis after a remote sacrocolpopexy carries significant morbidity. Mesh removal by means of minimally invasive surgery in the hands of an experienced surgical team utilizing DaVinci Robotic System is a good option and may lead to best patient outcomes.",book:{id:"11040",title:"Hysterectomy - Past, Present and Future",coverURL:"https://cdn.intechopen.com/books/images_new/11040.jpg"},signatures:"Adriana Fulginiti, Frank Borao, Martin Michalewski and Robert A. Graebe"},{id:"80782",title:"Cases of Postpartum Hemorrhage and Hysterectomy in Thailand’s Northern and Northeastern Provincial Hospitals",slug:"cases-of-postpartum-hemorrhage-and-hysterectomy-in-thailand-s-northern-and-northeastern-provincial-h",totalDownloads:31,totalDimensionsCites:0,doi:"10.5772/intechopen.102948",abstract:"PPH is a major cause of maternal death. Hysterectomy is safe to treat uncontrollable PPH. However, it may not be the best option for women who want to have children. The risk score tool to detect PPH earlier is needed in low-resource cities such as Chiang Rai and Sakon Nakhon province. This study aims to perform a risk score tool to prevent PPH in the northern and northeastern hospitals in Thailand; using mixed methods, identify risk factors for PPH from 20 articles globally and in Thailand using Med Calc, and develop the tool for prediction of PPH; and tool testing and a one-year follow-up on PPH-related hysterectomy cases. Results showed that this risk score tool can detect PPH earlier, reducing the number of PPH and hysterectomy cases. This risk score tool needs to be implemented in the same situations as hospitals to save pregnant women’s lives.",book:{id:"11040",title:"Hysterectomy - Past, Present and Future",coverURL:"https://cdn.intechopen.com/books/images_new/11040.jpg"},signatures:"Thawalsak Ratanasiri, Natakorn I. Tuporn, Somnuk Apiwantanagul, Thitima Nutrawong, Thawalrat Ratanasiri and Amornrat Ratanasiri"},{id:"80633",title:"Hysterectomy: Past, Present and Future",slug:"hysterectomy-past-present-and-future",totalDownloads:27,totalDimensionsCites:0,doi:"10.5772/intechopen.103086",abstract:"Hysterectomy is a major operation and is as old as time. This chapter touches briefly on the history of this procedure, its present aspects and general advice for these women who may need a hysterectomy, and finally the direction of new developments about it.",book:{id:"11040",title:"Hysterectomy - Past, Present and Future",coverURL:"https://cdn.intechopen.com/books/images_new/11040.jpg"},signatures:"Zouhair Odeh Amarin"},{id:"80589",title:"Total Vaginal Hysterectomy for Unprolapsed Uterus",slug:"total-vaginal-hysterectomy-for-unprolapsed-uterus",totalDownloads:54,totalDimensionsCites:0,doi:"10.5772/intechopen.101383",abstract:"Vaginal hysterectomy was the first method to extract the uterus. Vaginal hysterectomy goes back a long way into the history of medicine. Although the first hysterectomy was carried out by Themison of Athens in the year 20 B.C., the idea of extracting the uterus through the vagina was first mentioned in 120 B.C. by Soranus of Ephesos, a distinguished obstetrician. The first elective vaginal hysterectomy was performed by J. Conrad Langenbeck in 1813. The patient was a 50-year-old multipara, who suffered from chronic pelvic pain attributed to a prolapsed uterus with a hard, bleeding tumor. The operation was carried out in challenging conditions, without anesthesia, proper instruments, or surgical assistants. Until the early 1950s, vaginal hysterectomy was the method of choice for removing the uterus. With the widespread introduction of general anesthesia and antibiotic therapy, the site of vaginal hysterectomy was taken over by abdominal hysterectomy. With the introduction of minimally invasive surgery in gynecology, vaginal hysterectomy has regained its place. Harry Reich performed the first total laparoscopic hysterectomy in 1989, being one of the most renowned vaginal surgeons, and he still claims at the beginning of the 21st century that … when the first choice of approach for hysterectomy is possible, is the vaginal route. This chapter presents the relevant anatomy from the point of view of the vaginal surgeon and the standard technique used by the author in over 5,000 vaginal hysterectomies. All intraoperative drawings and photographs are original.",book:{id:"11040",title:"Hysterectomy - Past, Present and Future",coverURL:"https://cdn.intechopen.com/books/images_new/11040.jpg"},signatures:"Petre Bratila"},{id:"80400",title:"Laparoscopic Hysterectomy in Morbidly Obese Patients",slug:"laparoscopic-hysterectomy-in-morbidly-obese-patients",totalDownloads:33,totalDimensionsCites:0,doi:"10.5772/intechopen.101307",abstract:"The following chapter will focus on laparoscopic hysterectomy in morbidly obese patients. The discussion reviews the physiological changes associated with morbid obesity and the potential implications on pneumoperitoneum during laparoscopic surgery. Important considerations such as perioperative care and operating room setup are discussed. Additionally, obtaining abdominal access, reviewing the surgical approach, and post-operative considerations are all highlighted within this chapter.",book:{id:"11040",title:"Hysterectomy - Past, Present and Future",coverURL:"https://cdn.intechopen.com/books/images_new/11040.jpg"},signatures:"Merima Ruhotina, Annemieke Wilcox, Shabnam Kashani and Masoud Azodi"},{id:"80238",title:"Surgical Site Infection after Hysterectomy",slug:"surgical-site-infection-after-hysterectomy",totalDownloads:59,totalDimensionsCites:0,doi:"10.5772/intechopen.101492",abstract:"Surgical site infections (SSIs) are associated with increased morbidity, mortality, and healthcare costs. SSIs are defined as an infection that occurs after surgery in the part of the body where the surgery took place. Approximately 1–4% of hysterectomies are complicated by SSIs, with higher rates reported for abdominal hysterectomy. Over the past decade, there has been an increasing number of minimally invasive hysterectomies, in conjunction with a decrease in abdominal hysterectomies. The reasons behind this trend are multifactorial but are mainly rooted in the well-documented advantages of minimally invasive surgery. Multiple studies have demonstrated a marked decrease in morbidity and mortality with minimally invasive surgeries. Specifically, evidence supports lower rates of SSIs after laparoscopic hysterectomy when compared to abdominal hysterectomy. In fact, the American College of Obstetricians and Gynecologist recommends minimally invasive approaches to hysterectomy whenever feasible. This chapter will review the current literature on surgical site infection (SSI) after hysterectomy for benign indications.",book:{id:"11040",title:"Hysterectomy - Past, Present and Future",coverURL:"https://cdn.intechopen.com/books/images_new/11040.jpg"},signatures:"Catherine W. Chan and Michael L. 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",coverUrl:"https://cdn.intechopen.com/series/covers/22.jpg",latestPublicationDate:"May 18th, 2022",hasOnlineFirst:!0,numberOfPublishedBooks:1,editor:{id:"356540",title:"Prof.",name:"Taufiq",middleName:null,surname:"Choudhry",slug:"taufiq-choudhry",fullName:"Taufiq Choudhry",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0033Y000036X2hvQAC/Profile_Picture_2022-03-14T08:58:03.jpg",biography:"Prof. Choudhry holds a BSc degree in Economics from the University of Iowa, as well as a Masters and Ph.D. in Applied Economics from Clemson University, USA. In January 2006, he became a Professor of Finance at the University of Southampton Business School. He was previously a Professor of Finance at the University of Bradford Management School. He has over 80 articles published in international finance and economics journals. 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