5. Use of patients' sera for demonstrating the target pathogens in paraffin sections

Sera of patients suffering from infectious diseases expectedly contain high-titer IgG-type antibodies against the causative microbe, particularly when inflammatory reactions such as abscess and granuloma are histopathologically confirmed in immunocompetent individuals. It is well known that 2 weeks is needed to have specific antibodies to be raised in the serum. The diluted patients'serum can be used as a probe for indirect immunoperoxidase staining on histopathologic specimens routinely embedded in paraffin [4, 7, 8, 20–23]. A variety of infectious microbes were demonstrable with reliable sensitivity but limited specificity, as indicated below. Endogenous human immunoglobulins (IgG) in formalin-fixed,

Low-Specificity and High-Sensitivity Immunostaining for Demonstrating Pathogens… DOI: http://dx.doi.org/10.5772/intechopen.85055

paraffin-embedded sections were scarcely detected by the peroxidase-labeled secondary antihuman immunoglobulins. High-sensitivity detection sequence such as the labeled polymer method naturally leads to high-background staining, because of the detection of endogenous immunoglobulins in sections. The method is simple, economic, useful, and beautiful for the histopathologic diagnosis of infectious diseases, and it is particularly suitable for the developing countries, since the patient's serum is free in charge. This approach is especially effective for detecting protozoa and helminth [22], because the specific antibodies are often commercially unavailable.

There are two situations. In some cases, the causative pathogen has been identified by clinical, laboratory, and/or histopathological analysis, and thus the specificity of the patient's serum can be expected before immunostaining. In other cases, the causative pathogen is unsettled yet or unknown. In the latter situation, the patient's serum functions as a low-specificity and high-sensitivity probe in immunostaining.

Table 4 summarizes patients'sera applicable to immunostaining in paraffin sections.

#### 5.1 Methodology

Human tissues and organs, obtained either by biopsy, during surgery or at autopsy, were routinely fixed in 10% unbuffered or buffered formalin for 1 day to 4 weeks. The indirect immunoperoxidase technique was applied to deparaffinized sections [4, 7, 8, 20–23]. The serum of patient, principally not in an immunocompromised state, was diluted at 1:500 or 1:1000 and was incubated for 30 min or overnight. In case of protozoan infection, the presence of high-titer IgG antibodies in the patients'serum was commonly confirmed by immunofluorescence titration, as shown in Table 4. The serum from patients positive for human immunodeficiency virus (HIV) or hepatitis virus markers must not be utilized, in order to avoid biohazard. The serum of immunocompromised, non-HIV patients may be utilized after dilution at 1:5 or 1:10. The second layer reagent was horseradish peroxidase-labeled goat IgG to human immunoglobulins (Dako/Agilent) at a 1:50 dilution. Endogenous peroxidase activity was quenched in methanol containing 0.3% hydrogen peroxide for 20 min. No other pretreatment procedures such as proteinase digestion or heat-induced antigen retrieval were needed, but heating pretreatment was infrequently necessary for certain instances (e.g., the detection of free-living amoeba, Balamuthia; see below). Therefore, the author recommends introducing a heat-induced antigen retrieval procedure in 10 mM citrate buffer, pH 6, for immunostaining using silane (3-aminopropyltrimethoxysilane)-coated glass slides. After the diaminobenzidine coloring reaction, the nuclei were counterstained with 5% methyl green or Mayer's hematoxylin. When necessary, paraffin sections of related or unrelated infectious lesions were immunostained to confirm the cross-reactivity or specificity of the patients'serum.

It is of notice that IgG in the patients'serum may show cross-reactivity to related pathogens to certain or considerable degrees [4, 20, 21]. In bacterial and fungal infections, the sera often serve as pan-bacterial or pan-fungal probes. The crossreactivity may result from the naturally acquired antibodies in healthy individuals [20]. In viral, protozoan, and helminthic infections, in contrast, high-grade specificity with limited cross-reactivity can be expected [22]. When the specificity of the sera of patients with parasitic infestation is known, it is sufficiently satisfactory to enable them to be employed as specific antibody reagents for the following new cases.

#### 5.2 Immunostaining using sera of patients with established diagnosis

Here, the immunohistochemical application of sera of patients with established or fixed diagnosis is shown, including bacterial, fungal, viral, protozoan, and helminthic infections.

5. Use of patients' sera for demonstrating the target pathogens in

Lethal Pseudomonas aeruginosa pneumonia/septicemia (left, H&E; center, immunostaining using monoclonal antibody B11; right, immunostaining using B. cereus antiserum). Gram-negative bacilli grow along the alveolar septum, and no cellular reactions are noted. Immunoreactive signals are observed with the

The widest cross-reactivity shown by Bacillus cereus antiserum (left upper, brain abscess; right upper, ascending chorioamnionitis of placenta; lower panels, Propionibacterium acnes-induced folliculitis of the chest, combination of H&E, and B. cereus immunostaining). Gram-positive bacterial microbes are visualized by immunostaining with B. cereus antiserum. No positivity is seen with BCG, T. pallidum, and E. coli antisera.

Sera of patients suffering from infectious diseases expectedly contain high-titer IgG-type antibodies against the causative microbe, particularly when inflammatory reactions such as abscess and granuloma are histopathologically confirmed in immunocompetent individuals. It is well known that 2 weeks is needed to have specific antibodies to be raised in the serum. The diluted patients'serum can be used as a probe for indirect immunoperoxidase staining on histopathologic specimens routinely embedded in paraffin [4, 7, 8, 20–23]. A variety of infectious microbes were demonstrable with reliable sensitivity but limited specificity, as indicated below. Endogenous human immunoglobulins (IgG) in formalin-fixed,

paraffin sections

monoclonal antibody, as well as with B. cereus antiserum.

Immunohistochemistry - The Ageless Biotechnology

Figure 23.

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Figure 22.
