Detection of herpesvirus-like DNA by nested PCR on archival skin biopsy specimens of various forms of Kaposi sarcoma.

AIMS: To detect herpesvirus-like DNA sequences, defining a new herpesvirus, human herpesvirus 8 (HHV8), in paraffin wax embedded skin biopsy specimens of the various forms of Kaposi sarcoma. METHODS: DNA was extracted from archival skin biopsy specimens of Kaposi sarcoma, other mesenchymal skin tumours and various inflammatory skin lesions of HIV seropositive and negative patients. HHV8 DNA was detected by using a nested PCR assay. Human beta-globin DNA served as an internal control. RESULTS: Twenty two samples of Kaposi sarcoma were analysed, comprising 12 of the endemic type, nine HIV associated and one transplantation related. HHV8 DNA was detected by nested PCR in all forms of Kaposi sarcoma. By contrast, no HHV8 DNA was detected in five mesenchymal skin tumours or nine biopsy specimens of unspecific inflammatory skin lesions of HIV seropositive and negative patients. CONCLUSIONS: Detection of HHV8 DNA in paraffin wax embedded tissue can be used to confirm a diagnosis of Kaposi sarcoma.     

A Comparison of Lectin Binding to Frozen,Formalin Fixed Paraffin Embedded,and Acid Decalcified Paraffin Embedded Tissues

Abstract

A comparison was made between lectin binding to frozen, formalin fixed paraffin wax embedded, and to formic acid treated, paraffin wax embedded tissues, using three lectins UEA I, PNA, SBA. The binding to various rat tissues was assessed with a multiple layer immunoperoxidase staining method. Except for an increase in binding to some tissues, there was little difference between the acid treated and non-acid treated formalin fixed, paraffin embedded tissues. With several tissues, differences between frozen and paraffin embedded specimens suggested that care should be exercised in the choice of tissue processing if lectin histochemistry is to be undertaken. (The J Histotechnol 11:223, 1988.)     

Comparative analysis of biotin intranuclear inclusions of gestational endometrium using the APAAP, ABC and the PAP immunodetection systems.

AIMS/BACKGROUND: Intranuclear inclusions have been observed in gestational endometrial glands. Although resembling Herpes simplex virus infected cells, these nuclei have been shown to contain endogenous biotin. Hence, immunohistochemical systems using the avidin-biotin complex will result in cross reaction and false positivity. This study investigates a series of 10 gestational endometria (formalin fixed and paraffin wax embedded) with intranuclear inclusions using three different immunodetection systems with primary anti-biotin. RESULTS: Both the alkaline phosphatase anti-alkaline phosphatase (APAAP) and peroxidase anti-peroxidase (PAP) systems confirmed that the intranuclear inclusions were endogenous biotin. The streptavidin biotin complex (StreptABC) system produced a positive reaction in both test sections and controls (omitting primary anti-biotin) in the presence of prior blocking with free avidin and biotin. In addition, aberrant immunoreactivity was observed in adjacent nuclei/cytoplasm of endometrial glands and decidualised stroma in the negative control of the PAP system. This was subsequently eliminated using microwave pretreatment prior to immunohistochemistry. CONCLUSION: The use of either the APAAP or PAP immunodetection systems (the latter with microwave pretreatment) is recommended for any immunohistochemical or non-isotopic in situ hybridisation investigation undertaken on gestational endometria.     

Comparison of proliferation rates assessed using "multiblock" and conventional tissue blocks of lung carcinoma.

AIMS: To compare the proliferative rates, assessed immunohistochemically, of human lung tumours using conventional paraffin wax blocks and the multitumour tissue block (MTTB) technique. METHODS: A multiblock containing 20 lung tumours (eight adenocarcinomas, five squamous cell, five small cell and two carcinoid tumours) was constructed. Sections were also cut from the original blocks of formalin fixed, paraffin wax embedded tissue used to construct the multiblock. Sections were stained with the monoclonal antibody PC10, which recognises a proliferating cell nuclear antigen, using the three stage immunoperoxidase technique. RESULTS: The proliferation rates of the lung tumours obtained using both techniques were, overall, significantly different (p = 0.05), although most cases showed good correlation. Some tumours displayed a high degree of intratumoral variation in PC10 staining. The degree of PC10 staining was in keeping with the known proliferative state of particular histological subtypes--that is, carcinoid tumours showed little staining and small cell carcinomas showed extensive positivity. CONCLUSION: The MTTB technique is a less suitable means of assessing proliferation rate in lung carcinomas than conventional tissue blocks. It should be restricted to qualitative antibody studies or quantitative studies using tumours with little intratumoral variation.     

Use of the avidin-biotin peroxidase system to detect rabies antigen in formalin-fixed paraffin-embedded tissues

We stained rabies-infected nervous and salivary-gland tissues fixed in formalin or acetone and embedded in paraffin with the avidin-biotin peroxidase system. With this system, rabies-virus antigen was detected in neurons, glandular acinar cells, and vascular endothelial cells more effectively than by immunofluorescence, especially when tissues were enzyme-digested with pronase before immunoperoxidase staining. The avidin-biotin peroxidase system should be useful for routine diagnosis, retrospective studies of rabies, and identification of specific cells involved in the spread of virus in rabies-infected hosts.     

An alternative protocol for DNA extraction from formalin fixed and paraffin wax embedded tissue

BACKGROUND: DNA extraction from paraffin wax embedded tissue requires special protocols, and most described methods report an amplification success rate of 60-80%. AIMS: To propose a simple and inexpensive protocol consisting of xylene/ethanol dewaxing, followed by a kit based extraction. METHOD: Xylene/ethanol dewaxing was followed by a long rehydration step and a kit based DNA extraction step. RESULTS: This method produced a 100% amplification success rate for fragments of 121 to 227 bp for tamponated formalin fixed paraffin wax embedded tissue. CONCLUSION: This cost effective and non-laborious protocol can successfully extract DNA from tamponated formalin fixed paraffin wax embedded tissue and should facilitate the molecular analysis of a large number of archival specimens in retrospective studies.     

Detection of clonal B cells in microdissected reactive lymphoproliferations: possible diagnostic pitfalls in PCR analysis of immunoglobulin heavy chain gene rearrangement.

AIMS: To evaluate the specificity of standard and fluorescence based (Genescan) polymerase chain reaction (PCR) immunoglobulin heavy chain (IgH) gene rearrangement analysis in complete and microdissected paraffin wax embedded sections from lymphoid proliferations. METHODS: PCR IgH gene rearrangement analysis of whole sections and microdissected fragments (n = 62) from paraffin wax embedded reactive lymph nodes (n = 6) and tonsils (n = 3). Amplificant analysis used both standard methods and automated high resolution fluorescence based quantification and size determination using GENESCAN software. RESULTS: Whole tissue sections were consistently polyclonal in control experiments. IgH gene amplification was successful in 59 of 62 microdissected fragments; only two of 59 showed a polyclonal rearrangement pattern, the remainder being oligoclonal or monoclonal. Reanalysis was possible in 33 samples; six showed reproducible bands on gel analysis and satisfied accepted criteria for monoclonality. Use of high resolution gels with Genescan analysis improved sensitivity and band definition; however, three samples still appeared to be monoclonal. CONCLUSIONS: These results confirm that PCR based IgH gene rearrangement analysis is a sensitive and specific method for demonstrating B cell clonality in whole paraffin wax embedded sections. However, oligoclonal and monoclonal rearrangement patterns are regularly encountered in small tissue fragments from otherwise unremarkable reactive lymphoproliferations, possibly because of preferential priming or detection of local B cell clones. Data from clonal analysis of small, microdissected or lymphocyte poor samples must be evaluated critically. It is recommended that analyses should be run in parallel on at least two tissue specimens. Only reproducible bands present in more than one sample should be considered to be suggestive of neoplasia.     

Detection of clonal B cells in microdissected reactive lymphoproliferations: possible diagnostic pitfalls in PCR analysis of immunoglobulin heavy chain gene rearrangement.

AIMS: To evaluate the specificity of standard and fluorescence based (Genescan) polymerase chain reaction (PCR) immunoglobulin heavy chain (IgH) gene rearrangement analysis in complete and microdissected paraffin wax embedded sections from lymphoid proliferations. METHODS: PCR IgH gene rearrangement analysis of whole sections and microdissected fragments (n = 62) from paraffin wax embedded reactive lymph nodes (n = 6) and tonsils (n = 3). Amplificant analysis used both standard methods and automated high resolution fluorescence based quantification and size determination using GENESCAN software. RESULTS: Whole tissue sections were consistently polyclonal in control experiments. IgH gene amplification was successful in 59 of 62 microdissected fragments; only two of 59 showed a polyclonal rearrangement pattern, the remainder being oligoclonal or monoclonal. Reanalysis was possible in 33 samples; six showed reproducible bands on gel analysis and satisfied accepted criteria for monoclonality. Use of high resolution gels with Genescan analysis improved sensitivity and band definition; however, three samples still appeared to be monoclonal. CONCLUSIONS: These results confirm that PCR based IgH gene rearrangement analysis is a sensitive and specific method for demonstrating B cell clonality in whole paraffin wax embedded sections. However, oligoclonal and monoclonal rearrangement patterns are regularly encountered in small tissue fragments from otherwise unremarkable reactive lymphoproliferations, possibly because of preferential priming or detection of local B cell clones. Data from clonal analysis of small, microdissected or lymphocyte poor samples must be evaluated critically. It is recommended that analyses should be run in parallel on at least two tissue specimens. Only reproducible bands present in more than one sample should be considered to be suggestive of neoplasia.     

Immunoglobulin deposits in the biliary remnants of extrahepatic biliary atresia: a study by immunoperoxidase staining in 128 infants

Bile duct remnants taken from 128 infants during surgery for extrahepatic biliary atresia were paraffin embedded prior to immunoperoxidase staining. Immunoglobulin deposits were found in 44 remnants. They were made up of IgM alone in 25 cases and of IgM and IgG in 19. Deposits were observed along the basement membranes of glandular structures. These findings suggest that extrahepatic bilary atresia might be an acquired and evolving disease.     

A new mdr-1 encoded P-170 specific monoclonal antibody: (6/1C) on paraffin wax embedded tissue without pretreatment of sections.

AIMS: The generation and characterisation of a monoclonal antibody that specifically recognises the mdr-1 encoded protein, P-glycoprotein (P-170), on routinely processed formalin fixed, paraffin wax embedded tissue sections. METHODS: The monoclonal antibody, designated 6/1C, was produced following a combination of in vivo and in vitro immunisation regimens in Balb/c mice with a synthetic 12 amino acid peptide that corresponds to amino acids 21-32 (believed to be intracellularly located) of P-170 and has insignificant homology with the mdr-3 encoded P-170. Antibody 6/1C was characterised by western blotting and immunocytochemistry on cytospins of paired multidrug resistant or sensitive cell lines, including mdr-1 and mdr-3 transfected cells, and by immunohistochemistry on normal and malignant formalin fixed paraffin wax embedded tissue sections. RESULTS: Antibody 6/1C showed a single band at 170 kDa on western blots of multidrug resistant cell lysates and mdr-1 transfected cell lysates that was absent on similar preparations of drug sensitive cells and mdr-3 transfected cells. Immunocytochemical studies on cytospins of multidrug resistant cells and mdr-1 transfected cells revealed strong inner plasma membrane/cytoplasmic staining. Staining was negligible on drug sensitive cells and cells transfected with the mdr-3 gene. Immunohistochemical studies on formalin fixed, paraffin wax embedded normal adult kidney, liver, and breast tissue and a range of fetal tissues exhibited staining patterns of a variety of secretory surfaces consistent with documented mdr-1 specific staining. Specific staining of malignant cells in similarly treated sections of breast tumours was seen also with antibody 6/1C. Staining on paraffin wax embedded tissue with this antibody did not require any pretreatment of tissue sections. CONCLUSIONS: This new monoclonal antibody, chosen for its specificity with the mdr-1 encoded P-170 and its reactivity on routinely fixed paraffin wax embedded tissue samples without pretreatment, appears to be useful for the investigation of P-170 in archival material. It is especially useful for retrospective studies on pretreatment and post-treatment tissue sections, and could help establish when and how rapidly mdr-1 associated drug resistance develops during chemotherapeutic regimens. Immunohistochemical assessment of P-170 expression in many cancers has potential for diagnostic purposes and may influence the choice of chemotherapeutic drugs used in the treatment of refractory tumours.     

Immunoperoxidase staining for identification of Aspergillus species in routinely processed tissue sections.

AIMS: To evaluate the performance of an immunoperoxidase stain using the monoclonal antibody EB-A1 to detect Aspergillus species in formalin fixed, paraffin wax embedded tissue. METHODS: The monoclonal antibody EB-A1 directed against galactomannan was used to detect Aspergillus species in 23 patients with suspected or confirmed invasive aspergillosis. Immunostaining was performed on formalin fixed, paraffin wax embedded tissue using the streptavidin-biotin method and compared with conventional haematoxylin and eosin, periodic acid-Schiff, and Gomori-Grocott stains. Results of immunostaining were semiquantitatively analysed with regard to both intensity of staining and number of positively staining micro-organisms. Tissue sections from 16 patients with confirmed invasive mycoses due to Candida species, Apophysomyces elegans, Rhizopus oryzae, Pseudallescheria boydii and Histoplasma capsulatum were used as controls. RESULTS: In 19 (83%) of 23 cases invasive aspergillosis was confirmed by both histological examination and culture (18 Aspergillus fumigatus and one A flavus). Immunoperoxidase stains were positive in 17 (89%) of 19 cases including one case of disseminated infection due to A flavus. Furthermore, the immunoperoxidase stain was positive in a culture negative tissue section with histological evidence of mycelial development, indicating the presence of Aspergillus species. Some cross-reactivity was observed with the highly related fungus P boydii, although the number of mycelial elements that stained was low. CONCLUSIONS: Immunoperoxidase staining using the monoclonal antibody EB-A1 performs well on routinely processed tissue sections and permits detection and generic identification of Aspergillus species, although it was no better than conventional histopathology in identifying the presence of an infection. An additional advantage is that the immunostain may help to provide an aetiological diagnosis when cultures remain negative.     

Presence of 3-fucosyl-N-acetyllactosamine shown by monoclonal antibody AGF 4.48 in Reed-Sternberg cells.

A series of 50 specimens of Hodgkin's disease and 10 of reactive follicular hyperplasia were examined by means of indirect immunoperoxidase staining with a monoclonal antibody AGF 4.48: this is known to bind to 3-fucosyl-N-acetyllactosamine, which, in particular, is expressed by granulocyte series cells. Most Reed-Sternberg and many Hodgkin's cells were labelled by the antibody after pretreatment with neuraminidase. Routinely processed paraffin wax embedded sections proved suitable for staining. The findings were comparable with those reported by others with monoclonal antibodies to various other granulocyte markers. This technique is of potential diagnostic value.     

PCR detection of HIV proviral DNA (gag) in the brains of patients with AIDS: comparison between results using fresh frozen and paraffin wax embedded specimens.

AIMS--To adapt the polymerase chain reaction (PCR) technique of HIV detection to paraffin wax embedded brain tissue and to compare the results with those obtained using frozen tissue. METHODS--HIV antigen and HIV proviral DNA were detected in specimens of frontal lobe using immunohistochemistry and PCR, respectively. DNA was extracted from fresh tissue using standard methods whereas the technique for extracting DNA from paraffin wax embedded tissue was partly modified. RESULTS--Twenty cases were examined. HIV DNA was detected in 16 cases in frozen specimens. Of these, 15 were also positive when paraffin wax embedded material was analysed. CONCLUSIONS--This study shows that HIV proviral DNA can be detected in formalin fixed, paraffin wax embedded brain tissue by PCR. The results obtained from paraffin wax embedded specimens showed a similar degree of reliability to those from fresh frozen brain. Factors such as fixative, fixation time, and delay in performing post mortem examinations did not seem to influence PCR amplification as positive results were obtained with specimens left in fixative for up to eight months, as well as in cases where post mortem examinations had been delayed for up to four days.     

Does human papillomavirus play a role in the development of bladder transitional cell carcinoma? A comparison of PCR and immunohistochemical analysis

AIM: To investigate the role of human papillomavirus (HPV) in the development of bladder transitional cell carcinoma (TCC). METHODS: Seventy eight paraffin wax embedded TCC samples were tested for the presence of HPV by two methods. First, immunohistochemistry was carried out using a polyclonal antibody capable of detecting the capsid protein of all known papillomaviruses. The second method was a consensus GP5+/6+ primer mediated polymerase chain reaction (PCR) technique, with the products analysed by both agarose gel electrophoresis and an enzyme immunoassay using type specific oligonucleotide probes for 10 different mucosal genotypes. To exclude false negative results because of the poor quality of DNA extracted from paraffin wax embedded samples, the series was extended to include 20 further blocks for which the corresponding snap frozen unfixed tissue was available. RESULTS: The two methods produced contrasting results, with 47 of the 78 samples positive for HPV antigen and none positive for HPV DNA. HPV DNA was not detected in the 20 additional paraffin wax embedded TCCs or in the 20 paired unfixed samples. In contrast, HPV DNA was amplified by PCR from all six of the paraffin wax embedded cervical carcinoma and anogenital wart control samples. CONCLUSION: The disparity between the two sets of results is probably caused by false positives resulting from the non-specificity of the polyclonal antibody used for immunohistochemistry. These results suggest that HPV is unlikely to play an aetiological role in the development of bladder TCC.     

PCR analysis in archival postmortem tissues.

BACKGROUND: Formalin fixed and paraffin wax embedded tissues of necropsy origin are an important source for molecular analysis especially in rare diseases, neuropathology, or molecular epidemiology studies. Because of DNA degradation, only short sequences can be amplified from this type of tissue, very often less than 100 bases. This poses problems because studies on polymorphism and mutations occurring in large genes often require the analysis of long sequences. METHODS: The development of a simple treatment to obtain longer fragments of DNA for the analysis of archival postmortem paraffin wax embedded tissues. RESULTS: It was possible to amplify longer sequences ranging up to 300 bases from postmortem tissues, with no modification to the usual DNA extraction procedures. To obtain longer stretches of DNA, a pre-PCR restoration treatment was required, by filling single strand breaks, followed by a vigorous denaturation step. CONCLUSIONS: The development of this simple treatment allowed the analysis of longer fragments of DNA obtained from archival postmortem paraffin wax embedded tissues.     

Interphase cytogenetics using biotin and digoxigenin labelled probes: III. Increased sensitivity and flexibility for detecting HPV in cervical biopsy specimens and cell lines.

A monoclonal antibody to digoxin enabled sandwich techniques to be used for the detection of hybridised digoxigenin labelled probes in cultured cells and paraffin wax sections. This system has greater flexibility than alkaline phosphatase conjugated polyclonal antidigoxigenin antibody and permits the use of alternative detector enzymes, such as horseradish peroxidase and fluorescence labels. The APAAP detection system that does not require the use of biotin can also be used in situations where endogenous biotin is a problem. The low level of background staining combined with precise substrate deposition of the amplified peroxidase system gives higher sensitivity and resolution. This permits localisation of closely adjacent chromosomal loci in interphase nuclei. The most sensitive peroxidase based digoxigenin detection system visualises two and a half to 12 copies of human papillomavirus (HPV) per nucleus. This system is also suitable for the analysis of low copy number HPV infection of cervical tissues.     

Technical improvements in the immunoperoxidase study of renal biopsy specimens.

Sixty five renal biopsy specimens were used to compare a direct immunofluorescence technique on frozen sections with immunoperoxidase techniques on paraffin wax sections. For the immunoperoxidase techniques, dewaxed sections were treated with protease at 37 degrees C. Sections were examined at intervals on a microscope and digestion was stopped when plasma was removed from glomerular capillary loops. This permitted intense staining of immunoproteins on immunoperoxidase. There was agreement between immunoperoxidase and immunofluorescence in the staining for IgG, IgA, and IgM in 50 biopsy specimens and discordant findings did not affect the diagnosis. Immunoperoxidase did not detect C3 in 16 biopsy specimens. Findings with antiserum to another complement component, C9, detected by immunoperoxidase correlated with C3 findings detected by immunofluorescence in 17 biopsy specimens. It is concluded that microscopical observation of the progress of digestion permits optimal staining by immunoperoxidase methods, thus overcoming the problem of variability in proteolytic digestion of sections. Inconsistency in the demonstration of complement deposition can be avoided by staining for C9 rather than C3.     

Comparison of in situ methods to assess DNA cleavage in apoptotic cells in patients with breast cancer.

BACKGROUND: Apoptosis has a role in many cellular processes including development, normal tissue homeostasis, and malignancy. This aspect of research is relatively new with distinct methods of analysing disparate biochemical and genetic events to measure apoptotic cells. The use of biotinylated nucleotides to identify DNA strand breaks is a commonly reported method of estimating cells numbers undergoing apoptosis; however, investigators report inconsistent results for a variety of reasons. AIMS AND METHOD: To compare two in situ techniques of measuring apoptosis: in situ nick translation (ISNT) and TdT mediated dUTP-biotin nick end labelling (TUNEL); and to assess DNA cleavage in 20 paired paraffin wax embedded breast cancer tissues from patients; one group who had received no prior treatment and one group who had received chemohormonal treatment. RESULTS AND CONCLUSIONS: Apoptotic scores obtained from paraffin wax embedded human breast cancer after using ISNT and TUNEL methods were not significantly different (p = 0.11). A strong correlation between scores obtained from the two techniques was found (r = 0.758, p < 0.0001). Optimisation of both techniques is crucial to ensure maximal assay performance in breast cancer tissue.     

Immunohistological detection of human cytotoxic/suppressor T cells using antibodies to a CD8 peptide sequence.

AIMS: To evaluate whether cytotoxic/suppressor T cells can be detected in paraffin wax embedded human tissue samples using antibodies to a synthetic CD8 peptide sequence. METHODS: Polyclonal and monoclonal antibodies were raised against a 13 amino acid peptide sequence from the cytoplasmic portion of the alpha chain of the human CD8 molecule. RESULTS: These antibodies specifically detected the native form of the CD8 polypeptide when tested by immunoprecipitation with radiolabelled T cells, and gave the expected staining pattern for cytotoxic/suppressor T cells in cryostat sections. Being raised in rabbits, the polyclonal antibodies were also useful for double labelling for CD8 in conjunction with monoclonal antibodies. CD8 positive cells could also be detected in paraffin wax embedded tissues. This was achieved without prior treatment of the sections if the tissue had been fixed in Bouin's fixative. When tissues had been exposed to conventional formalin fixation, preliminary microwave treatment was required. CONCLUSIONS: These findings provide further evidence that antibodies against leucocyte associated antigens, capable of reacting on paraffin wax embedded tissue, can be produced by immunisation with synthetic peptide sequences.