Technologies Used To Confirm Histopathologic Diagnosis in Dermatopathology
World Small Animal Veterinary Association World Congress Proceedings, 2004
Verena K. Affolter
School of Veterinary Medicine, University of California-Davis
Davis, CA, USA

Conventional methods

 Cultures: 1) fungal cultures from hair shafts, scrapings, aspirates and biopsies; 2) bacterial cultures from aspirates, sterile biopsies; 3) testing for antibiotic resistance.

 Smears from aspirates

 Biopsy: 1) hematoxylin and eosin (H&E) stain: morphologic features; 2) special stains: infectious pathogens, structures, inflammatory cells, deposition of extracellular matrix.

 Electron microscopy: EM is mainly used for ultra-structural evaluations of tissues and the identification of small pathogens such as viruses. Prompt collection and proper fixation (Karnovsky's solution; glutaraldehyde) are important as ultra-structural anatomy is easily obscured by autolysis. However, often formaldehyde fixed tissue can still be used for EM.

The exploding world of "CD's"

The access to monoclonal antibodies (MAB) has led to the identification of many cell surface and cytoplasmic molecules (antigens: proteins, glycoproteins, etc.). These MAB are an invaluable tool for the characterization of the structure, molecular weight and function of many antigens.

Because MAB technology is such a powerful tool, many laboratories began making MAB (for example MAB specific for leukocytic antigens). As would be expected, many laboratories used slightly different protocols for naming and characterization of these MAB. These efforts resulted in a very confusing situation where many different names were used for in fact one single molecule (NU-TH/1, LEU3a, B14, RPA-T4 and OKT4 represent anti-CD4 antibodies). Workshops were organized in order to resolve the confusion of different names and characterizations of leukocyte antigens and antibodies; first "Human Leukocyte Differentiation Antigen Workshop" was completed in 1982. Many different laboratories were involved in testing the antibodies (immunohistochemistry on lymphoid tissue, flow cytometry on blood samples, immunoprecipitation/Western blots). The result was the assignment of a "cluster of differentiation" (CD) for each specific antigen evaluated. Similar workshops were organized for assigning CD designations in other species (sheep, pig, dog, and horse).

The use of antibodies

 Panel of MAB: Most antigens--in particular leukocytic antigens--are not lineage specific. Therefore, a leukocytic cell population cannot be reliably identified by using one single MAB. Tumor cells may change their expression of some surface molecules or down regulate the expression of others. This emphasizes, that tumor cells always have to be evaluated by a panel of relevant MAB.

 Species-specific differences in expression of antigens and cross-reactivity: The distribution of some antigens vary markedly between species. Cross-reactivity of a particular MAB has to be thoroughly evaluated before the antibody can be used reliably in another species.

 Phenotypic studies

 Immunohistochemistry or immunofluorescence to evaluate cells in tissue sections, cells in smears of aspirates, cytospins or imprints

 Flow cytometry for quantitative evaluation of cells in solution: blood, aspirates, solubilized tumor cell populations

 ELISA for evaluation of presence of specific antibodies in a serum

 Immunoprecipitation/Western blot: Identification of an antigen by its molecular weight

 Functional studies: Presence of certain antigens indicates functional status of a cell population. Moreover, by binding to a particular epitope, some MAB can activate cells or, alternatively, block function of the cells

 Limitation of use of MAB: For most instances clonality (presence of a clonal versus a polyclonal cell population) cannot be evaluated by using MAB (see clonality studies below).

Immunohistochemistry (IHC) and immunofluorescence (IF)

At times morphology and cytology are not sufficient to identify cells, especially when the cells are neoplastic and have lost normal characteristics. With the use of MAB specific molecules on the surface and in the cytoplasm are identified; this process allows identifying the cell origin. IHC and IF also allow us to identify certain structures in the tissue, which are not apparent in conventional stains. The biggest advantage of IHC is the possibility to identify specific antigens within the context of a tissue sample. Hence, this requires that during the process the tissue antigens remain their antigenicity and at the same time, the tissue architecture is maintained. The evaluation of IHC is mostly a qualitative one. Quantitative evaluations are not accurate for the most part. Flow cytometry offers a far more accurate tool to do quantitative evaluations of cell populations. IHC uses an enzymatic reaction and a chromogen. With IF the antibodies are labeled with a fluorescent dye (examples: fluorescence isothiocyanate (FITC) or Texas red (TR); light source: mercury lamp).

IHC on formalin-fixed and paraffin-embedded tissues: Formalin-fixation allows good maintenance of tissue and cell morphology. However, formalin forms hydroxymethylene bridges between protein chains (cross-linking). This results in change of epitopes, which then may be lost for detection by a specific MAB. Some of these cross-linkings can be reversed by a process called antigen retrieval. This process includes either enzymatic digestion (trypsin, pronase, pepsin), boiling or steaming in citric acid or a combination thereof. The tissue sections are collected on "sticky" slides to prevent detachment of the tissue during the fairly long procedure.

Cryosections from fresh-frozen tissues: Tissue samples can be collected and kept fresh by wrapping them in saline dampened gauze, and putting them in zip lock bags or small pill containers to be shipped on a cooling package. Fresh tissue samples are snap frozen in an OCT-compound without previous fixation to assure maintenance of the native stage of the epitopes of the antigens. The morphology of frozen samples is not as good as with formalin-fixed, paraffin-embedded samples. The tissues are collected on "sticky" slides to prevent detachment of the tissue during the fairly long procedure.

Cytospins, impressions and smears from aspirates are air dried and then processed like frozen sections.

Controls are essential to interpret the results reliably and to determine whether the positive staining represents a genuine positive result or is non-specific.

Association with morphologic features on hematoxylin-eosine stained tissue sections--Immunohistochemistry is always an additional tool to conventional morphologic pathology. The findings in immunohistochemistry have to be carefully correlated with the morphology of the lesions.

Applications: 1) identification of skin structures: basement membrane components; 2) identification of cel populations: round cell tumors, spindle cell tumors; 3) deposition of immunoglobulins and complement: immune-mediated diseases; 4) identification of infectious pathogens: example BCG stain

Immuno-electron microscopy (IEM)

Antibodies can also be coupled with small gold particles and subsequently used on samples prepared for EM. The localization of the gold particles (bound by specific antibodies) can then be visualized with EM.

Flow cytometry

The technology of flow cytometry and fluorescence activated cell sorting (FACS) was developed in the early 1970's. Living cells in solution are evaluated for their reactivity with fluorescence-coupled reagents; examples: fluorescence isothiocyanate (FITC), phycoerythrin (PE); light source: UV light. While passing through a tube, the cells are excited by a laser beam. Cells labeled with fluorescent dye emit light after excitement. This emitted light is registered as a positive signal. Flow cytometry is a convenient and fast procedure. Advantages of flow cytometry includes that heterogeneous cell populations can easily be evaluated and different sub-populations can be quantitated. Double labeling with two different antibodies is easier to interpret than with immunohistochemistry. However, the cells are evaluated individually and out of their context. So there is no information about their relation to surrounding tissue.

Samples: Single cell suspensions

Applications: 1) immunophenotyping of cell populations in aspirates: tumor cell populations in ascites, pleural and pericardial effusions. 2) immunophenotyping of peripheral blood cells: Identification of leukemia, 3) follow-up evaluation of leukemias: monitoring of therapy success, residual diseases recurrence, 4) evaluation of DNA content of cells: identification of the stage of cell cycle (cells in G2 phase have a double amount of DNA).

Analysis: Data received through acquisition are saved and stored. The results can be accessed and analyzed later. The data can be analyzed quantitatively and statistic analysis can be performed.

Detection of a protein by its molecular weight: Immunoprecipitation and immunoblotting/Western blotting.

Immunoprecipitations with subsequent immunoblotting or Western blots have been developed to identify proteins based on their molecular weight.

Immunoprecipitation with subsequent immunoblotting: Labeled solubilized proteins are detected and precipitated by specific immobilized antibodies. The antibody-captured antigens are separated according to their molecular weight over an electrophoresis gel. The proteins are then transferred to a membrane by transverse electrophoresis. The size of the protein can subsequently be visualized comparing with molecular weight markers; depending on the label by an enzyme reaction or chemiluminescence on radiographic films.

Western blotting: The solubilized antigen mixture is first separated by their molecular weight by gel electrophoresis and the proteins are transferred to a membrane. Antigens of interest are then detected by labeled specific antibodies. The remaining procedure is similar to immunoprecipitation and blotting.

Samples: patient's serum and tissue samples

Applications: 1) identification of autoantibodies in patients sera (antibody source: patient's serum, antigen source: normal tissue of same species or synthetic peptides): diagnosis of auto-immune diseases and immune-mediated diseases, 2) identification of presence of certain tissue proteins (antigen source: patients tissues, antibody source: commercial specific antibodies): diagnosis of congenital diseases involving deficiency of certain proteins

Enzyme-linked immunoabsorbent assay (ELISA)

Either antigens or antibodies are linked to a plastic surface. With fixed antigens, the presence of antibodies can be evaluated. With fixed antibodies the presence of antigens can be evaluated by a second antibody step (sandwich method). The antibodies are tagged with an enzyme and the intensity of the color reaction--after adding substrate--reflects the concentration of the antigen or antibody. When compared with a standard curve (produced by known protein concentrations) the concentration of the antibody/antigen can be determined. This is for example used for antibodies titration in serum samples.

Samples: Patents serum, tissue lysates from patients

Applications: 1) identification of presence of specific antibodies in a patient serum (plate coated with antigens, antibodies in the serum): antibodies against pathogens; auto-antibodies, titration of antibodies, 2) identification of the presence of an antigen (plate is coated with antibodies, antigen is added from patients tissues): presence of pathogens in aspirates, isotyping of antibodies in patients serum (evaluation of IgE titers)

Radioimmunoassay (RIA)

Same principle as ELISA. The antibodies have a radioactive label.

Molecular techniques

The access to molecular technologies and recombinant DNA technologies had a dramatic impact on biology. Discovery of genes, new means to determine functions of proteins and of individual domains within proteins are achieved on a daily base with these technologies. Cloning, sequencing and polymerase chain reaction (PCR) have become helpful tools in research and daily diagnostics. However, the results from these procedures need to be interpreted in context of additional findings (clinical presentation, pathologic findings, immunophenotyping and clinical follow-up). Good positive and negative controls are crucial to avoid misinterpretation.

Isolation of DNA and RNA: Genomic DNA (gDNA) is very stable and can be isolated from tissues (formalin-fixed and fresh tissues), cell pellets, peripheral blood etc. gDNA is composed of exons and introns. Only exons code for the translation into mRNA, introns are cut out in the translation process. As RNA is fare less stable (RNA-digesting enzymes are ubiquitous); the isolated RNA is therefore immediately transferred into DNA using the enzyme reverse transcriptase. The recombinant DNA (cDNA) is complementary to RNA and is only composed of exons. Occasionally, RNA is preferred for some techniques such as in situ hybridization.

Samples: Fresh snap frozen tissues, aspirates, air-dried smears are excellent sources for DNA and RNA. Formalin-fixed, paraffin embedded tissues sections can be used for most reactions as well.

A. Polymerase chain reactions (PCR): The polymerase chain reaction allows amplification of minuscule amounts of DNA. If the sample of interest is RNA (example: identification of mRNA for certain proteins. RNA viruses), the template is first transcribed into cDNA. As little as DNA from one particular cell can be detected. Because such small amounts of DNA can be amplified the results must be interpreted in context with the positive and negative controls as well as with other procedures.
DNA is amplified by the enzyme polymerases, which read each strand of the template DNA from the 5'end towards the 3'end. DNA is double stranded, so a primer pair for each strand has to be designed according to the sequence of the DNA fragment of interest. With each cycle of PCR, the DNA segments between these primer templates (section where the primers anneal to) will be replicated. The end products from PCR are separated on an agarose gel, which identifies the length of the amplified DNA. The bands, representing the amplified DNA of interest, can be cut out from the gel and the DNA can be used for other procedures (see below: cloning, sequencing).

Applications: 1) identification of extra-cellular and intracellular pathogens: intra-lesional virus, partially degraded bacteria, 2) identification of mRNA for a certain protein: lack/presence of certain structural proteins, enzymes, cytokines, 3) clonality testing of cel populations: clonal or polyclonal infiltrates of T cells or B cells. Each T cell rearranges the T cell receptor genes and has therefore a unique segment of DNA, the variable region of the TCR. This can be used to determine whether a population of T cells is clonal and ergo has the exact same variable region, or if it is polyclonal and hence has different variable regions. The same procedure can be used for B lymphocytes, because they rearrange the V region of their B cell receptors /immunoglobulins.

B. In situ hybridization: Radioactive or enzyme labeled probes (small segments of DNA or RNA) are directly applied to tissue samples. The presence of DNA or RNA of interest (pathogens, virus, bacteria, cytokines, hormones, enzymes etc) can be identified in the context of the tissue sample and a particular cell population. In situ hybridization can be combined with immunohistochemistry. A disadvantage of in situ hybridization: very small amounts of RNA and DNA may not give enough signals to be visualize without amplification.

Applications: 1) identification of extracellular and intracellular pathogens: virus, parasites, mycobacteria, 2) identification of mRNA for a certain proteins: enzymes, cytokines, structural proteins

C. Alternative procedures are Southern blots to detect DNA and Northern blots to detect RNA. These procedures are more labor intensive and used more for research purposes than diagnostics.

Speaker Information
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Verena K. Affolter, DVM, DECVP, PhD
School of Veterinary Medicine, University of California-Davis
Davis, CA

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