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PubMed1992

Immunohistological detection of human parvovirus B19 in formalin-fixed, paraffin-embedded tissues.

Human parvovirus B19 is a cause of aplastic crises in patients with haemolytic anaemias, prolonged bone marrow failure in the immunosuppressed, and fetal death secondary to non-immune hydrops. The immunohistological detection of parvovirus B19 in formalin-fixed, paraffin-embedded tissues has not previously been reported, and definitive diagnosis of infection in such specimens has relied on the use of specialized DNA hybridization and amplification techniques. A new monoclonal antibody to B19 capsid proteins, R92F6, was found to be capable of labelling infected cells in paraffin-embedded tissues from all 19 cases of parvovirus-related fetal hydrops tested, and in bone marrow from a child with congenital immunodeficiency and chronic parvovirus infection. Viral antigen was detected both in cytoplasmic and in nuclear distributions using the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique without preceding proteolytic digestion. The viral epitope recognized appears to be highly conserved, as specimens were obtained over a 13-year period from widely spaced locations in the U.K. Antibody R92F6 should facilitate rapid diagnosis of parvovirus B19 infection in routinely processed and archival specimens.

Antibodies, Viral↗

PubMed2003

Detection of ALK gene rearrangements in formalin-fixed, paraffin-embedded tissue using a fluorescence in situ hybridization (FISH) probe: a search for optimum conditions of tissue archiving and preparation for FISH.

BACKGROUND: It is widely known that the efficiency of fluorescence in situ hybridization (FISH) probes applied to formalin-fixed, paraffin-embedded tissues is affected by the conditions under which the tissues are fixed and embedded. However, relatively few studies address exactly how tissue archiving conditions affect the performance of FISH probes. We report our experience based on use of an ALK FISH probe, during the validation of its diagnostic utility. METHODS: We applied the probe to 77 formalin-fixed, paraffin-embedded tissue blocks archived from 1991 through to 2000, and studied the interrelationship between the archival age (which ranged up to 10 years), type and condition of tissue, duration required for optimum hydrolysis, and obtainability of hybridization signals. RESULTS: We found that as archival age and tissue collagen content increased, not only did hydrolysis times have to be prolonged in order to yield interpretable hybridization signals, but also the likelihood of blocks becoming non-signaling increased. The most striking positive correlations were seen between the archival age of signaling lymphoid blocks and their requisite hydrolysis times. CONCLUSIONS: The difficulty in applying FISH on archival tissue increases with its archival age and collagen content, and may necessitate changes in laboratory protocol accordingly.

Anaplastic Lymphoma Kinase↗

PubMed1993

Comparison of two antibodies for evaluation of estrogen receptors in paraffin-embedded tumors.

Estrogen receptor (ER) content in breast cancer specimens is correlated with a prolonged disease free survival and increased likelihood of response to hormone therapy. Relatively few anti-ER antibodies are currently available for use in formalin-fixed, paraffin-embedded tissue. Recently, a new anti-ER monoclonal antibody (ERID5; AMAC, Westbrook, Maine) was generated which requires antigen retrieval by microwave oven heating for detection in routinely processed tissue. The specific aim of this study was to compare the ERID5 antibody with the commercially available rat monoclonal (ER-ICA; Abbott, Chicago, IL) which requires proteolytic enzyme digestion for detection in paraffin-embedded tissue. Sections from 20 cases of primary breast carcinoma previously assayed by dextran-coated charcoal (DCC) analysis were examined. Quantitation of ER antibody staining was performed without knowledge of the DCC values. Specimens containing > or = 20% specifically stained malignant cells were considered ER positive. The sensitivity and specificity of visual ER-ICA immunostaining were 57% and 83%, respectively. The sensitivity and specificity of visual ERID5 immunostaining were 93% and 50%, respectively. The predictive value of positive staining was 89% for the ER-ICA antibody and 81% for the ERID5 antibody. The predictive value of negative staining was 45% for the ER-ICA antibody and 75% for the ERID5 antibody. Previous studies have demonstrated a linear correlation between DCC values and the positive nuclear area (PNA) generated by image analysis for ER-ICA immunostaining. In the present study, a similar correlation between DCC value and ERID5 percentage PNA was observed (R = 0.670; P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

The effect of different fixatives and length of fixation time on subsequent AgNOR staining for frozen and paraffin-embedded tissue sections.

The AgNOR technique has been used extensively in studies investigating the possibility that the numbers and appearances of the intranuclear structures stained are markers of malignancy. The method has the advantage of being applicable to many different types of histological material, including paraffin-embedded tissue. However, it has been suggested that the visualization of AgNORs is dependent on the type and time of fixation employed. This study set out to measure this effect with the following commonly-used fixatives: acetone, absolute ethanol, methanol, Carnoy's fluid, Bouin's fluid, 4% glutaraldehyde, 10% neutral buffered formalin and 10% formol-saline. Both frozen sections and blocks of fresh tonsil were fixed for varying times, the blocks of tissue then being processed routinely. With the frozen sections AgNORs were easier to discern than in sections of paraffin-embedded tissue, and more intranucleolar AgNORs were visible when alcoholic fixatives were used than with aldehyde fixation. The effects of different fixatives on AgNOR appearance in paraffin sections is, however, more complex. Despite the variation caused by different fixatives, AgNORs could be demonstrated adequately with all the fixatives studied. It is concluded that fixation is not a limitation to the study of AgNORs provided that the time and type of fixative is controlled.

Acetates↗

PubMed1994

[Detection of B-cell clonality in paraffin-embedded bone marrow biopsy specimens by polymerase chain reaction].

In order to estimate the clonality of B-cell lymphoproliferative disorders, polymerase chain reaction (PCR) was used to detect the Ig heavy chain gene rearrangement in DNA extracted from formalin-fixed and paraffin-embedded bone marrow biopsies. 16 out of 19 cases (84%) of B-CLL (n = 9), multiple myeloma (n = 7) and B-non-Hodgkin's lymphoma (n = 3) showed sharp monoclonal bands, while polyclonal smears or no PCR-products were observed in non-neoplastic bone marrow or AML specimens. The results showed that the DNA from paraffin-embedded bone marrow biopsies could be used to detect monoclonal IgH rearrangement by PCR method. This forms the basis for the studies of minimal residual disease or B-ML bone marrow infiltrated diseases.

Biopsy↗

PubMed1990

Inter-laboratory comparison of DNA flow cytometric results from paraffin-embedded breast carcinomas.

Consecutive sections from 33 paraffin-embedded human breast carcinomas without intratumor heterogeneity were sent for flow cytometric (FCM) DNA analysis in two experienced laboratories. FCM instruments, run conditions, and tumor disaggregation procedures were different in the two laboratories. In four cases (12%) the laboratories reported a different DNA ploidy and DNA index (DI). These variations were due to analytical reasons, differences in the detection rates of near-diploid and tetraploid DIs, not due to interpretation of data or the criteria used for aneuploidy. There was a significant correlation between S-phase fractions (SPF) obtained in the two laboratories (r = 0.90, p less than 0.0001) if only cases with concordant DI were included. Discordant DI usually led to very different SPF values.

Aneuploidy↗

PubMed1995

Extramedullary myeloid cell tumors. An immunohistochemical study of 29 cases using routinely fixed and processed paraffin-embedded tissue sections.

OBJECTIVE: Extramedullary myeloid cell tumors (EMCTs) may be unsuspected clinically and difficult to recognize histologically. Fresh or frozen tissue is often not available for analysis. We studied 29 cases of EMCT using routinely fixed and processed paraffin-embedded tissue, an immunohistochemical method, and a panel of antibodies. PATIENTS: We studied 29 patients with EMCTs: 22 males and 7 females, with a median age of 48 years (range, 5 to 80 years). Histologically, 9 tumors were well differentiated, 16 were poorly differentiated, and 4 were blastic. RESULTS: The Leder stain (napthol-ASD-chloroacetate esterase) was positive in 21 (77.7%) of 27 tumors. Immunohistochemically, the following antibodies reacted with the greatest number of cases: Leu-22 or MT1 (CD43) in 28 (96.6%) of 29, antilysozyme in 27 (96.4%) of 28, and antimyeloperoxidase (MP07) in 21 (91.3%) of 23 cases. Other myeloid lineage-associated antibodies were positive in a subset of cases: antineutrophil elastase (NP57) in 10 (62.5%) of 16, Leu-M1 (CD15) in 7 (46.6%) of 15, and Mac-387 in 6 (40.0%) of 15 cases. The well-differentiated EMCTs reacted with most myeloid-associated antibodies; poorly differentiated and blastic tumors were more often negative. The pan-leukocyte antibody LCA (CD45RB) reacted with 15 (60%) of 25 neoplasms. Three (16.6%) of 18 tumors contained numerous p53-positive cells, ranging from 10% to 50% of the tumor cell population. In 10 cases, exons 5 through 8 of the p53 gene were analyzed using the polymerase chain reaction and single-stranded conformational polymorphism analysis. Gel shifts consistent with mutations were identified in exon 8 of one tumor (10%) that exhibited abundant p53 immunostaining. CONCLUSIONS: Immunohistochemical studies using fixed, paraffin-embedded sections are very useful in the diagnosis of EMCTs. The most sensitive antibodies are anti-CD43, antilysozyme, and antimyeloperoxidase. Immunohistochemical methods are more sensitive than the Leder stain. We found p53 staining in a small subset of cases, in which we were able to confirm evidence of p53 gene mutation using the polymerase chain reaction and single-stranded conformational polymorphism analysis in one case; p53 gene mutations appear to be uncommon in EMCTs.

Adult↗

PubMed2004

Molecular typing of Pneumocystis jirovecii found in formalin-fixed paraffin-embedded lung tissue sections from sudden infant death victims.

Previous studies have provided histological evidence of an association between primary Pneumocystis infection and sudden infant death syndrome (SIDS). The aim of this work was to determine the species of clustered Pneumocystis organisms found in formalin-fixed paraffin-embedded (FFPE) lung tissue sections from Chilean sudden infant death (SID) victims. This approach needed first to optimize a DNA extraction method from such histological sections. For that purpose, the QIAamp DNA Isolation from Paraffin-Embedded Tissue method (Qiagen) was first tested on FFPE lung tissue sections of immunosuppressed Wistar rats inoculated with rat-derived PNEUMOCYSTIS: Successful DNA extraction was assessed by the amplification of a 346 bp fragment of the mitochondrial large subunit rRNA gene of the Pneumocystis species using a previously described PCR assay. PCR products were analysed by direct sequencing and sequences corresponding to Pneumocystis carinii were found in all the samples. This method was then applied to FFPE lung tissue sections from Chilean SID victims. Pneumocystis jirovecii was successfully identified in the three tested samples. In conclusion, an efficient protocol for isolating PCR-ready DNA from FFPE lung tissue sections was developed. It established that the Pneumocystis species found in the lungs of Chilean SID victims was P. jirovecii.

Animals↗

PubMed1986

Immunohistologic detection of estrogen receptors in paraffin-embedded breast cancers: correlation with cytosol measurements.

Paraffin-embedded sections of 76 human breast tissue specimens were analyzed for estrogen receptors (ER) and endogenous bound estrogen (ER-E). Preincubation of sections with polyestradiol phosphate was followed by stabilization of the complex with glutaraldehyde. The bound hormone was then visualized by the peroxidase-antiperoxidase (PAP) technique with antiestradiol as the primary antiserum. Normal breast tissue and benign proliferations were consistently positive for ER and ER-E. All specimens were examined for free and bound receptors in cytoplasm and nuclei. Among the carcinomas examined, a high correlation was found between the presence of ER by the PAP method and by the biochemical analysis of cytosol preparations. The PAP method, requiring no special preparation of surgical specimens, overcomes many of the disadvantages of the cytosol method and adds the advantage of independent evaluation of nuclear and cytoplasmic estrogen binding sites.

Breast Neoplasms↗

PubMed2005

Improved detection of mycobacterial DNA by PCR in formalin-fixed, paraffin-embedded tissues using thin sections.

PCR is a unique methodology allowing for the sensitive detection of mycobacterial DNA-sequences in cases in which no fresh material can be obtained for classic analyses. Despite the limitations of this technique, for example the less satisfactory quality of DNA from paraffin-embedded specimens and the high effort necessary to control contamination, PCR still represents a useful additional tool for routine diagnostic examinations of mycobacterial infections. Fragmentation of the DNA extracted from formalin-fixed, paraffin-embedded samples on the one hand and the rigid cell wall of mycobacteria on the other hand are obstacles to detecting the DNA of these microorganisms by PCR. Here, we describe a simple mechanical procedure that allows us to improve the detection of mycobacterial DNA with the use of thin (1 microm) sections instead of thicker sections. This could be explained by a gentle, mechanical opening of the acid fast mycobacterial cell wall. Thus, even the application of heat/cold shock treatments is not necessary. This inexpensive fast procedure can also be used for the detection of other infectious agents.

DNA, Bacterial↗

PubMed1989

A novel monoclonal antibody (OPD4) recognizing a helper/inducer T cell subset. Its application to paraffin-embedded tissues.

A novel monoclonal antibody (MAb), OPD4, reactive with a helper/inducer (H/I) subset of T cells in formalin-fixed, paraffin-embedded tissue sections, has been identified through immunization with an activated H/I T cell line, namely DL40. The antibody is an IgG1 antibody and it recognizes an antigen with a molecular weight of 200 kd, corresponding to that of leukocyte common antigen. OPD4+/CD4+ T cells provided better help for pokeweed mitogen-stimulated polyclonal IgG production than OPD4-/CD4+ T cells. OPD4 recognized the H/I T cell subset even in paraffin-embedded tissue sections, but did not recognize nonhematopoietic cells, suppressor/cytotoxic T cells, B cells, monocytes in the peripheral blood, or other normal hematopoietic cells as examined by the flow cytometric and immunoperoxidase methods. Besides the lymphoid cells, OPD4 reacted with a number of histiocytes (epithelioid cells) in tissues from sarcoidosis and tuberculosis. For the neoplastic lesions, OPD4 reacted with approximately half of the cases of T cell lymphomas. Consequently, OPD4 may be useful for the diagnosis and study of malignant lymphomas and other related lesions.

Antibodies, Monoclonal↗

PubMed1994

Chemical dehydration for rapid paraffin embedding.

We describe chemical dehydration with 2,2-dimethoxypropane (DMP) for rapid paraffin embedding using a mixture of DMP and mineral oil followed by mineral oil as clearing intermediates. This method is useful for classical histological techniques as well as for histochemistry and immunocytochemistry.

Desiccation↗

PubMed1987

Practical importance of routine paraffin-embedded bone marrow biopsy in multiple myeloma.

Paraffin-embedded bone marrow biopsy specimens obtained prior to (37) or after (25) therapy from 62 patients with multiple myeloma (MM) were analyzed with particular reference to infiltration pattern, extent of infiltration, and myeloid to myeloma tissue percentage ratio (MMR) to verify their mutual relationships and clinicopathologic relevance. Fifty-nine biopsies were evaluable for infiltration pattern (diffuse in 27, interstitial in 25, and nodular in 7). Diffuse and interstitial patterns were more common (P less than 0.025) in stage III and stage I patients, respectively. A higher (P less than 0.001) mean serum paraprotein level was found in patients with the diffuse pattern than in those with the interstitial pattern. The average extent of infiltration by myeloma cells in the residual myeloid tissue was higher (P less than 0.001) and a high extent (75% or more) was more frequently (P less than 0.005) seen in diffuse than in interstitial pattern cases. The average MMR value was lower (P less than 0.001) and a MMR value less than 1 was more frequently (P less than 0.005) seen in the diffuse pattern group than in the interstitial pattern group. All these differences were present also when a separate analysis was performed for treated and untreated patients. It seems that a diffuse histologic pattern, as opposed to interstitial, would significantly predict a bone marrow extent of infiltration of 75% or more, a MMR lower than 1, a higher serum paraprotein level, and a clinical stage III. Bone marrow biopsy appears thus to play a role in providing parameters of prognostic relevance in MM also in the course of the disease. Prospective studies are needed to establish whether histologic pattern has an independent prognostic value.

Biopsy↗

PubMed2005

Exposure of nuclear antigens in formalin-fixed, paraffin-embedded necropsy human spinal cord tissue: detection of NeuN.

Immunohistochemical and immunofluorescence staining approaches are powerful tools for characterization of the endogenous protein expression and subcellular compartmentalization. However, several technical problems hamper identification of low-abundance nuclear proteins in archival formalin-fixed, paraffin-embedded human neural tissue. These include loss of protein antigenicity during tissue fixation and processing, and intrinsic auto-fluorescence associated with the tissue related to its fixation and the presence of lipofuscin. We evaluated several antigen retrieval methods to establish a strategy for detection of neuronal nuclear proteins in human spinal cord formalin-fixed, paraffin-embedded tissue. Thus, using immunostaining of the neuron-specific nuclear protein NeuN as the outcome measure, we found that heating tissue sections in an alkaline pH buffer unmasked protein epitopes most effectively. Moreover, staining by immunohistochemistry with diaminobenzidine tetrahydrochloride chromagen was superior to immunofluorescence labeling, likely due to the signal amplification steps included in the former approach. Auto-fluorescence in the tissue sections can be effectively reduced, but a sufficient fluorescence signal associated with specific antibody labeling could not be detected above this background for NeuN in the nucleus.

Animals↗

PubMed2006

Glioma test array for use with formalin-fixed, paraffin-embedded tissue: array comparative genomic hybridization correlates with loss of heterozygosity and fluorescence in situ hybridization.

Array-based comparative genomic hybridization (aCGH) is a powerful, high-throughput tool for whole genome analysis. Until recently, aCGH could only be reproducibly performed on frozen tissue samples and with significant tissue amounts. For brain tumors however, paraffin-embedded tissue blocks from small stereotactic biopsies may be the only tissue routinely available. The development of methods to analyze formalin-fixed, paraffin-embedded (FFPE) material therefore has the potential to impact molecular diagnosis in a significant way. To this end, we constructed a BAC array representing chromosomes 1, 7, 19, and X because 1p/19q deletion and EGFR gene amplification provide clinically relevant information for glioma diagnosis. We also optimized a two-step labeling procedure using an amine-modified nucleotide for generating aCGH probes. Using this approach, we analyzed a series of 28 FFPE oligodendroglial tumors for alterations of chromosomes 1, 7, and 19. We also independently assayed these tumors for 1p/19q deletion by fluorescence in situ hybridization and by loss of heterozygosity analyses. The concordance between aCGH, standard loss of heterozygosity and fluorescence in situ hybridization was nearly 100% for the chromosomes analyzed. These results suggest that aCGH could offer an improved molecular diagnostic approach for gliomas because of its ability to detect clinically relevant molecular alterations in small FFPE specimens.

Cold Temperature↗

PubMed1989

Flow cytometry using paraffin-embedded tissue: five years on.

The use of paraffin-embedded tissue for flow cytometry is reviewed. A number of technical modifications of the original 1983 method have been described, aimed at improving the accuracy of DNA measurements by minimizing cell debris or reducing coefficients of variation, and at simplifying sample preparation. Over 100 clinical studies have now been reported, mainly assessing the effect of DNA index on prognosis, and those published up until mid-1988 are summarized in an appendix. More recently there have been developments in the use of monoclonal antibodies to measure oncogene products or proliferation markers in addition to DNA content. Detailed clinical evaluation and standardization of these more sophisticated methods is still some way ahead, but as was the case with DNA index, the use of archival material from patients whose outcome is already known should speed this process.

Animals↗

PubMed1996

Chromosomal translocation t(X;18) in human synovial sarcomas analyzed by fluorescence in situ hybridization using paraffin-embedded tissue.

Synovial sarcoma is characterized cytogenetically by translocation t(X;18)(p11.2;q11.2). In this study, 28 cases that had been diagnosed initially as synovial sarcoma, including 2 fibrosarcomas, and 1 leiomyosarcoma were collected and examined for translocation t(X;18) on paraffin-embedded tissues by fluorescence in situ hybridization (FISH). Of the synovial sarcomas, 25 showed findings consistent with translocation t(X;18) with an additional copy signal for the total probe of X and 18 chromosomes. The other three cases, as well as the two fibrosarcomas and the leiomyosarcoma, did not show this translocation. One (case 26) of three negative cases was diagnosed finally as leiomyosarcoma and another (case 27) as malignant peripheral nerve sheath tumor from histological and immunohistochemical analysis. Thus, in all, 25 (96%) of 26 synovial sarcomas showed findings consistent with translocation t(X;18). In summary, translocation t(X;18) is a chromosomal aberration specific for synovial sarcoma. The fluorescence in situ hybridization technique can be used even on cells from paraffin-embedded tissues, and is a useful diagnostic aid for synovial sarcoma.

Adolescent↗

PubMed1994

Detection of the 4977 base pair mitochondrial DNA deletion in paraffin-embedded heart tissue using the polymerase chain reaction--a new method to probe sudden cardiac death molecular mechanisms?

Detection of mitochondrial DNA deletions is performed in fresh or frozen material. At our institute, however, heart samples from subjects referred for autopsy are systematically processed for histologic examination (that is, paraffin-embedded). We were interested to know if mtDNA deletions can be detected in such material. Our data indicate that: 1) the most frequently observed deletion--the 4977 base pair deletion--can easily be detected in paraffin-embedded heart tissue; 2) this assay is sufficiently sensitive, since very low levels of the deletion can be found in normal heart tissue from young adults; and 3) buffered formalin appears to be the fixative of choice. Recent literature shows that repeated episodes of ischemia result in the accumulation of mtDNA deletions in myocardial cells. Because ischemic heart disease is a major cause of sudden cardiac death, a sensitive method for the detection of mtDNA damage in myocardial cells will be an important tool to facilitate understanding of unexpected cardiac arrest mechanisms.

Adolescent↗