José A. Ramos-Vara

Technical Aspects of Immunohistochemistry

Immunohistochemistry is an integral technique in many veterinary laboratories for diagnostic and research purposes. In the last decade, the ability to detect antigens (Ags) in tissue sections has improved dramatically, mainly by countering the deleterious effects of formaldehyde with antigen retrieval (AR) and increasing sensitivity of the detection systems. In this review, I address these topics and provide an overview of technical aspects of immunohistochemistry, including those related to antibodies (Abs) and Ags, fixation, AR, detection methods, background, and troubleshooting. Microarray technology and the use of rabbit monoclonal Abs in immunohistochemistry are also discussed.

Retrospective Study of 338 Canine Oral Melanomas with Clinical, Histologic, and Immunohistochemical Review of 129 Cases

Diagnostic records from 338 canine oral melanomas in 338 dogs received at the Veterinary Medical Diagnostic Laboratory (1992-1999) were reviewed. Of these tumors, 122 plus an additional 7 metastatic melanomas of unknown origin were selected for clinical follow-up, histologic review, and immunohistochemistry. Chow Chow, Golden Retriever, and Pekingese/Poodle mix breeds were overrepresented, whereas Boxer and German Shepherd breeds were underrepresented. There was no gender predisposition and the average age at presentation was 11.4 years. Forty-nine dogs were euthanized due to recurrence or metastasis. The average postsurgical survival time was 173 days. The gingiva and the labial mucosa were the most common sites. Most tumors were composed of either polygonal cells (27 cases, 20.9%), spindle cells (44 cases, 34.1%), or a mixture of the two (polygonal and spindle) (54 cases, 41.9%). Clear cell (3 cases, 2.3%) and adenoid/papillary (1 case, 0.8%) patterns were uncommon. The metastases of 6/6 oral melanomas had morphologic and immunohistochemical features similar to those of the primary tumors. Immunohistochemically, Melan A was detected in 113/122 oral (92.6%) and 5/7 (71.9%) metastatic melanomas. Only 4/163 nonmelanocytic tumors were focally and weakly positive for Melan A. Antibodies against vimentin, S100 protein, and neuron-specific enolase stained 129 (100%), 98 (76%), and 115 (89.1%) of 129 melanomas, respectively. Antibodies against other melanocyticassociated antigens (tyrosinase, glycoprotein 100) did not yield adequate staining. We conclude that Melan A is a specific and sensitive marker for canine melanomas.

Identification of porcine circovirus in tissues of pigs with porcine dermatitis and nephropathy syndrome

Thirty-three pigs affected by porcine dermatitis and nephropathy syndrome, 30 from Spain and three from the USA, were investigated in order to detect porcine circovirus (Pcv) in their tissues. A standard in situ hybridisation technique using a specific DNA 317-bp probe based on a well-conserved sequence of Pcv (which recognises both Pcv-i and PCV-2) was applied to formalin-fixed, paraffin-embedded tissues. Twentyeight of the 30 Spanish pigs and all three American pigs had Pcv in at least one tissue. Viral nucleic acid was detected mainly in lymphoid organs, and especially the lymph nodes. The viral genome was also found, in order of decreasing quantity, in Peyers patches, tonsil, lung, spleen, kidney, liver, and skin. Viral nucleic acid was located mainly within the cytoplasm of monocyte/macrophage lineage cells, including follicular dendritic cells, macrophages, histiocytes and Kupffer cells. No viral nucleic acid was found in damaged glomeruli or arteriolar walls. In frozen samples available from three Spanish pigs, the virus was identified as type 2 by using the polymerase chain reaction and restriction fragment length polymorphism. Most of the pigs from which serum was available were seropositive against porcine respiratory and reproductive syndrome virus (PRRSV), and PRRSV antigen was detected in the lung of two of the Spanish pigs. These results suggested that Pcv is present in tissues of almost all pigs affected by PDNS, and Pcv has to be considered as a possible agent involved in the pathogenesis of the syndrome.

Proposal of a 2-Tier Histologic Grading System for Canine Cutaneous Mast Cell Tumors to More Accurately Predict Biological Behavior

Currently, prognostic and therapeutic determinations for canine cutaneous mast cell tumors (MCTs) are primarily based on histologic grade. However, the use of different grading systems by veterinary pathologists and institutional modifications make the prognostic value of histologic grading highly questionable. To evaluate the consistency of microscopic grading among veterinary pathologists and the prognostic significance of the Patnaik grading system, 95 cutaneous MCTs from 95 dogs were graded in a blinded study by 28 veterinary pathologists from 16 institutions. Concordance among veterinary pathologists was 75% for the diagnosis of grade 3 MCTs and less than 64% for the diagnosis of grade 1 and 2 MCTs. To improve concordance among pathologists and to provide better prognostic significance, a 2-tier histologic grading system was devised. The diagnosis of high-grade MCTs is based on the presence of any one of the following criteria: at least 7 mitotic figures in 10 high-power fields (hpf); at least 3 multinucleated (3 or more nuclei) cells in 10 hpf; at least 3 bizarre nuclei in 10 hpf; karyomegaly (ie, nuclear diameters of at least 10% of neoplastic cells vary by at least two-fold). Fields with the highest mitotic activity or with the highest degree of anisokaryosis were selected to assess the different parameters. According to the novel grading system, high-grade MCTs were significantly associated with shorter time to metastasis or new tumor development, and with shorter survival time. The median survival time was less than 4 months for high-grade MCTs but more than 2 years for low-grade MCTs.

Classification of Canine Malignant Lymphomas According to the World Health Organization Criteria

A study was carried out to test the accuracy and consistency of veterinary pathologists, not specialists in hematopathology, in applying the World Health Organization (WHO) system of classification of canine lymphomas. This study represents an initiative of the ACVP Oncology Committee, and the classification has been endorsed by the World Small Animal Veterinary Association (WASVA). Tissue biopsies from cases of canine lymphoma were received from veterinary oncologists, and a study by pathologists given only signalment was carried out on 300 cases. Twenty pathologists reviewed these 300 cases with each required to choose a diagnosis from a list of 43 B and T cell lymphomas. Three of the 20 were hematopathologists who determined the consensus diagnosis for each case. The 17 who formed the test group were experienced but not specialists in hematopathology, and most were diplomates of the American or European Colleges of Veterinary Pathology. The overall accuracy of the 17 pathologists on the 300 cases was 83%. When the analysis was limited to the 6 most common diagnoses, containing 80% of all cases, accuracy rose to 87%. In a test of reproducibility enabled by reintroducing 5% of cases entered under a different identity, the overall agreement between the first and second diagnosis ranged from 40 to 87%. The statistical review included 43,000 data points for each of the 20 pathologists.

Suggested guidelines for immunohistochemical techniques in veterinary diagnostic laboratories.

This document is the consensus of the American Association of Veterinary Laboratory Diagnosticians (AAVLD) Subcommittee on Standardization of Immunohistochemistry on a set of guidelines for immunohistochemistry (IHC) testing in veterinary laboratories. Immunohistochemistry is a powerful ancillary methodology frequently used in many veterinary laboratories for both diagnostic and research purposes. However, neither standardization nor validation of IHC tests has been completely achieved in veterinary medicine. This document addresses both issues. Topics covered include antibody selection, fixation, antigen retrieval, antibody incubation, antibody dilutions, tissue and reagent controls, buffers, and detection systems. The validation of an IHC test is addressed for both infectious diseases and neoplastic processes. In addition, storage and handling of IHC reagents, interpretation, quality control and assurance, and troubleshooting are also discussed. Proper standardization and validation of IHC will improve the quality of diagnostics in veterinary laboratories.

Optimization of immunohistochemical methods using two different antigen retrieval methods on formalin-fixed paraffin-embedded tissues: experience with 63 markers.

Formalin fixation produces cross-links between the proteins and the fixative that alter the ability of some antibodies to recognize antigens. We used formalin-fixed, paraffin-embedded tissues to compare two different antigen retrieval methods for 63 antibodies used in the diagnosis of infectious and neoplastic diseases of animal species. Eighty-four percent of the antibodies needed some type of antigen retrieval for optimal results. Of those antibodies, 67.7% were monoclonal and 32.3% were polyclonal. Steam heat was the method of choice for 31 antibodies. Ten antibodies reacted only with steam heat, but 9 antibodies did not react when steam heat was used. Optimal results were obtained with enzyme digestion for 22 antibodies. Only 10 antibodies yielded optimal results without antigen retrieval; 64% of these antibodies were polyclonal. All antibodies against cytokeratins were optimally retrieved with proteinese K. Antigen retrieval appears to be necessary for the majority of antibodies when used with formalin-fixed, paraffin-embedded tissues.

When Tissue Antigens and Antibodies Get Along Revisiting the Technical Aspects of Immunohistochemistry—The Red, Brown, and Blue Technique

Once focused mainly on the characterization of neoplasms, immunohistochemistry (IHC) today is used in the investigation of a broad range of disease processes with applications in diagnosis, prognostication, therapeutic decisions to tailor treatment to an individual patient, and investigations into the pathogenesis of disease. This review addresses the technical aspects of immunohistochemistry (and, to a lesser extent, immunocytochemistry) with attention to the antigen-antibody reaction, optimal fixation techniques, tissue processing considerations, antigen retrieval methods, detection systems, selection and use of an autostainer, standardization and validation of IHC tests, preparation of proper tissue and reagent controls, tissue microarrays and other high-throughput systems, quality assurance/quality control measures, interpretation of the IHC reaction, and reporting of results. It is now more important than ever, with these sophisticated applications, to standardize the entire IHC process from tissue collection through interpretation and reporting to minimize variability among laboratories and to facilitate quantification and interlaboratory comparison of IHC results.

Lipid profiles of canine invasive transitional cell carcinoma of the urinary bladder and adjacent normal tissue by desorption electrospray ionization imaging mass spectrometry.

Desorption electrospray ionization mass spectrometry (DESI-MS) was used in an imaging mode to interrogate the lipid profiles of thin tissue sections of canine spontaneous invasive transitional cell carcinoma of the urinary bladder (a model of human invasive bladder cancer) as well as adjacent normal tissue from four different dogs. The glycerophospholipids and sphingolipids that appear as intense signals in both the negative ion and positive ion modes were identified by tandem mass spectrometry product ion scans using collision-induced dissociation. Differences in the relative distributions of the lipid species were present between the tumor and adjacent normal tissue in both the negative and positive ion modes. DESI-MS images showing the spatial distributions of particular glycerophospholipids, sphinoglipids, and free fatty acids in both the negative and positive ion modes were compared to serial tissue sections that were stained with hematoxylin and eosin (H&E). Increased absolute and relative intensities for at least five different glycerophospholipids and three free fatty acids in the negative ion mode and at least four different lipid species in the positive ion mode were seen in the tumor region of the samples in all four dogs. In addition, one sphingolipid species exhibited increased signal intensity in the positive ion mode in normal tissue relative to the diseased tissue. Principal component analysis was also used to generate unsupervised statistical images from the negative ion mode data, and these images are in excellent agreement with the DESI images obtained from the selected ions and also the H&E-stained tissue.

Uterine neoplasia in 13 cats.

Thirteen uterine tumors were diagnosed in 13 cats and accounted for 0.29% of all feline neoplasms received during a 9.6-year period. Age at diagnosis ranged from 3 to 16 years; median 9 years. Six were Domestic Shorthair cats, and 7 were purebred cats of 5 different breeds. Eight adenocarcinomas and 1 mixed Müllerian tumor (adenosarcoma) comprised the endometrial tumors. Myometrial tumors included 3 leiomyomas and 1 leiomyosarcoma. One of the adenocarcinomas developed in the uterine stump of an ovario-hysterectomized cat; the other cats were sexually intact. Concurrent mammary adenocarcinoma was diagnosed in 1 cat with uterine adenocarcinoma and in another with uterine leiomyoma. Tumors were discovered during elective ovariohysterectomy in 2 cats, but at least 3 others had experienced reproductive problems (infertility or pyometra). Five cats presented for abdominal or pelvic masses. Endometrial adenocarcinomas were positive immunohistochemically for cytokeratins and negative for smooth muscle actin (SMA); 1 of 6 cats was positive for vimentin and 4 of 8 were positive for estrogen receptor—α (ERα). Adenosarcoma stromal cells were positive for vimentin and ERα but negative for cytokeratins and SMA. Smooth muscle tumors were positive for vimentin and SMA and negative for cytokeratins. Leiomyomas, but not the leiomyosarcomas, were positive for ERα. Adenocarcinomas in 4 cats had metastasized by the time of ovariohysterectomy. Two other cats were euthanized 5 months after ovariohysterectomy; at least one of these cats had developed an abdominal mass that was not examined histologically. Only 2 cats with endometrial adenocarcinoma had disease-free intervals longer than 5 months after surgery. Metastasis was not detected in any mesenchymal tumor; however, these cats were either euthanized on discovery of the tumor or the tumor was first detected at necropsy.