Demonstration of microRNA using in situ hybridisation on formalin fixed paraffin wax samples using conventional oligonucleotide probes: a comparison with the use of locked nucleic acid probes.

BACKGROUND: MicroRNAs (miRNAs) regulate the translation of mRNA during gene expression and investigations have highlighted their importance in pathophysiology. qRT-PCR is currently the gold standard method for detecting changes in miRNA expression. However, when used on heterogeneous samples, it cannot identify individual cell types harbouring miRNAs. For this, in situ hybridisation (ISH) can be used. ISH methods using locked nucleic acid (LNA) probes give reliable results in formalin fixed paraffin-embedded (FFPE) samples. In this study their use has been directly compared with conventional oligonucleotide probes (COP) for ISH. METHODS: FFPE samples of colorectal adenocarcinoma, squamous carcinoma of lung and cases of invasive breast carcinoma were used to evaluate COP and LNA methods for the demonstration of miR-126 and miR-205. To demonstrate the utility of the COP method demonstration of miR-21 in 19 Gleason stage 7 prostate biopsy FFPE tissues was also undertaken. The demonstration of miR-21 by ISH in high and low expressing prostate cancer cell lines was also compared with qRT-PCR. RESULTS: Similar results were obtained using the COP and LNA ISH methods for the demonstration of miR-126 and miR-205. miR-21 was successfully demonstrated in the prostate cancer samples by COP ISH and expression levels of the miRNA demonstrated in the cell lines corresponded with qRT-PCR. CONCLUSION: This study has shown that simplification of ISH protocols by the use of COPs provides equivalent results to the use of LNA methods and it can be used to precisely identify cells in which miRNAs are expressed.

Assessing the potential of immunohistochemistry for systematic gene expression profiling.

Immunohistochemistry (IHC) is a powerful technique for identifying sites of protein expression in tissues at the cellular and sub-cellular level. Here we have investigated the potential of using IHC for genome-wide expression screening by measuring the success rate and specificity of a panel of 35 monoclonal antibodies recognizing 5 well characterised CD antigens. Antibodies were pre-screened on acetone fixed frozen sections of spleen, tonsil and colon tissues. 19/35 antibodies gave staining with a success rate of 0/7 for JAM-2, 1/4 for CD99, 3/6 for CD138, 5/8 for CD45 and 10/10 for MHC-class II. 16/19 of these antibodies also gave staining on formalin fixed paraffin embedded tissue sections of tonsil and colon. All antibodies that had given staining were then profiled on tissues presented in human tissue microarrays. In the frozen microarrays 216 cores from 29 normal tissue types were present and in the formalin fixed paraffin array 344 cores from 35 normal and 4 cancers were represented. Where multiple antibodies were positive, there was evidence of consistent staining of the same tissues with several antibodies. In some cases differences in staining were observed potentially due to differential splice variants, polymorphisms or protein modification. With some antibodies there was evidence of cross-reactivity to inappropriate cells or structures. In addition the staining intensity with formalin fixation was changed quantitatively for some antibodies and in a few cases qualitatively, representing differential sensitivity of specific and non-specific epitopes to fixation. Accordingly, whilst IHC has potential for describing protein expression of unknown genes, these results emphasise a need to systematically address issues of specificity and sensitivity if appropriate profiles are to be described.

Cross-reactivity and clinical impact of the antibody response to hepatitis C virus second envelope glycoprotein (E2).

The genotype of hepatitis C virus (HCV) can profoundly affect the success of antiviral therapy for HCV infection. A possible contributing factor is a varied immune response elicited by infection with different HCV genotypes. In this study, full-length E2 proteins of HCV genotypes 1a, 1b, 2a, and 2b were used to determine the fraction of the humoral immune response to HCV E2 that is genotype specific. Greater than 90% of all infected individuals had serum antibodies to the four E2 proteins. Overall, individuals infected with genotype 1a or 1b were characterized by variable immune responses to HCV E2 with relatively high amounts of cross-reactivity with other E2 proteins. Individuals infected with genotype 2a or 2b exhibited a strong preferential reactivity to genotype 2a and 2b E2 proteins. Individuals with elevated titers to HCV E2 were more likely to be infected with genotype 2a and had a significantly lower median viral load. These findings indicate that the antibody response to HCV E2 is affected by the genotype of the virus and that induction of a strong humoral immune response to HCV E2 may contribute to a decreased viral load.

High degree of interlaboratory reproducibility of human immunodeficiency virus type 1 protease and reverse transcriptase sequencing of plasma samples from heavily treated patients.

We assessed the reproducibility of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and protease sequencing using cryopreserved plasma aliquots obtained from 46 heavily treated HIV-1-infected individuals in two laboratories using dideoxynucleotide sequencing. The rates of complete sequence concordance between the two laboratories were 99.1% for the protease sequence and 99.0% for the RT sequence. Approximately 90% of the discordances were partial, defined as one laboratory detecting a mixture and the second laboratory detecting only one of the mixture's components. Only 0.1% of the nucleotides were completely discordant between the two laboratories, and these were significantly more likely to occur in plasma samples with lower plasma HIV-1 RNA levels. Nucleotide mixtures were detected at approximately 1% of the nucleotide positions, and in every case in which one laboratory detected a mixture, the second laboratory either detected the same mixture or detected one of the mixture's components. The high rate of concordance in detecting mixtures and the fact that most discordances between the two laboratories were partial suggest that most discordances were caused by variation in sampling of the HIV-1 quasispecies by PCR rather than by technical errors in the sequencing process itself.

Isolation and tissue profiles of a large panel of phage antibodies binding to the human adipocyte cell surface.

Phage display is a powerful technique for the rapid selection and isolation of antibodies to any given target antigen. We have applied this technology to isolate over 100 different human antibodies that bind to antigens expressed in situ on the human adipocyte cell surface. This is a diverse panel of antibodies, as indicated by the V-region sequences. The binding profile of each anti-adipocyte antibody has been characterised using phage antibody immunocytochemistry against a panel of normal human tissues. Although there was some variation in the intensity of the adipocyte staining, each antibody consistently recognised adipocytes, where present, irrespective of the tissue source. In addition, all of the antibodies recognised at least one other cell type other than the adipocyte cell surface. In total, over 50 different tissue-binding profiles were recorded, with the most frequently recognised tissues identified as capillaries or smooth muscle. Extensive tissue binding profiles were generated for some antibodies using a panel of 37 different human tissues. This identified anti-adipocyte antibodies with unexpected profiles, such as FAT.13, which binds only to adipocytes and capillaries in the entire tissue panel. We believe this is the most extensive survey ever undertaken of the human adipocyte cell surface. Moreover, similar methodology could be used to derive complete tissue-binding profiles of antibodies against cell-surface antigens of any cell type. Indeed, by screening antibodies on both normal and diseased tissues, it may be possible to identify antigenic associations between different cell types and the pathologies of many diseases.

Reproducibility of human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase sequencing of plasma samples from heavily treated HIV-1-infected individuals.

The reproducibility of population-based human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase (RT) sequencing was assessed using replicate aliquots of cryopreserved plasma samples obtained from seven heavily treated HIV-1-infected individuals. The sequence of each sample replicate was compared with the consensus sequence for that sample and 99.4% of 35128 amino acids were found to be concordant with the sample consensus. Partial discordances were present at 0.5% of positions and complete discordances were present at <0.1% of positions. To assess the reproducibility at detecting mutations (defined here as differences from the subtype B consensus sequence), the proportion of sequences having a mutation when at least two sequences from that sample had the same mutation were examined. There was a median of 13 protease and 18 RT mutations per sample for a total of 3126 mutations; 95% of these mutations were detected. However, sequencing of multiple clones from two samples demonstrated that those mutations present in a minority of clones were often not detected by population-based sequencing. These results suggest that HIV-1 protease and RT sequencing of circulating plasma virus is highly reproducible but that the sensitivity at detecting mutations may be low if those mutations are present as minor variants.

HIV-1 genotypic resistance patterns predict response to saquinavir-ritonavir therapy in patients in whom previous protease inhibitor therapy had failed.

BACKGROUND: Tests for resistance to HIV drugs are available for clinical use; however, their predictive value has not been fully assessed. OBJECTIVES: To determine HIV-1 genotypic predictors of a virologic response to saquinavir-ritonavir therapy in patients in whom at least one previous protease inhibitor-containing regimen had failed and to compare the predictive value of baseline genotype with that of standard clinical evaluation. DESIGN: Retrospective clinical cohort study. SETTING: University-based HIV clinic. PATIENTS: 54 HIV-1-infected adults treated with saquinavir-ritonavir who had experienced virologic failure while receiving a protease inhibitor-containing regimen for at least 3 months. MEASUREMENTS: HIV-1 reverse transcriptase and protease gene sequences, CD4 cell counts, clinical characteristics, detailed antiretroviral treatment history, and plasma HIV-1 RNA levels at baseline and at three follow-up time points (median, 4, 12, and 26 weeks). Virologic failure was defined as a plasma HIV RNA level greater than 1000 copies/mL. RESULTS: In 22 patients (41%), a plasma HIV-1 RNA level less than 500 copies/mL was achieved by week 12; in 15 patients (28%), this response was maintained through week 26. Clinical characteristics predicting a poorer response included a diagnosis of AIDS, lower CD4 cell count, and higher plasma HIV RNA level (P<0.03). Number of previous nucleoside reverse transcriptase inhibitors, previous protease inhibitor therapy, and duration of previous protease inhibitor therapy were predictors of poorer response (P<0.01). Multivariate regression models revealed that protease mutations present at the initiation of saquinavir-ritonavir therapy were the strongest predictors of virologic response. A model of clinical features explained up to 45% of the variation in virologic outcomes by week 12, whereas the explained variance was 71% when genotypic predictors were included. CONCLUSIONS: In patients in whom protease inhibitor-containing antiretroviral therapy fails, HIV-1 genotype is predictive of virologic response to subsequent therapy. This predictive capacity adds to that of standard clinical evaluation.

Cytomegalovirus gB genotype distribution differs in human immunodeficiency virus-infected patients and immunocompromised allograft recipients.

Cytomegalovirus isolates can be grouped into 4 gB and 2 gH genotypes. gB genotypes were studied in patients infected with human immunodeficiency virus (HIV) and in allograft transplantation recipients. In allograft recipients, the distribution of gB 1, -2, -3, and -4 in leukocytes and urine, respectively, was 36%, 21%, 43%, and 0% and 39%, 30%, 17%, and 13%. However, in leukocytes of HIV-infected patients with <100/microL CD4 cells, gB1 was found significantly less often than in allograft recipients (11% vs. 36%) but gB2 was more frequent (56% vs. 21%; P < .05). The decreased incidence of gBl and increased incidence of gB2 compared with allograft recipients was also seen in urine of HIV-infected patients and reflected the distribution seen in leukocytes. gB4 was found significantly more often (P < .05) in semen than in leukocytes of HIV-infected patients with < 100/microL CD4 cells. gB1-4 genotypes were similar in patients with < 100/microL CD4 cells with or without retinitis.

The glycosylphosphatidylinositol-anchored lymphocyte antigen CDw52 is associated with the epididymal maturation of human spermatozoa.

The CAMPATH-1 (CDw52) antigen is a small glycosylphosphatidylinositol (GPI) anchored glycoprotein with a mature peptide comprising only 12 amino acids. It is abundantly expressed on human lymphocytes and is an unusually good target for complement-mediated cell lysis. The immunosuppressive and lymphocyte-depleting effects of CAMPATH-1 antibodies are being tested in a variety of diseases. Here we show that the antigen is also expressed at a high level in the male reproductive system, being found in the epididymis, seminal vesicle, seminal plasma and on the surface of mature (but not testicular) spermatozoa. Its possible transfer from epithelial cells in the epididymis to maturing sperm may represent a novel method of acquisition of cell surface antigens. In the presence of human complement, CAMPATH-1 antibodies inhibited the motility of washed sperm. However, seminal plasma blocks antibody binding and can protect sperm from this cytotoxic effect.

Direct detection of Molluscum contagiosum virus in clinical specimens by in situ hybridization using biotinylated probe.

Molluscum contagiosum virus (MCV) is an unclassified poxvirus which has recently become recognized as causing a major sexually transmitted disease. At present no assay is available for specific detection of MCV because the virus cannot be serially propagated in cell culture. Since MCV produces an abortive, limited growth with some cytopathic effect in certain cell lines, we were able to develop an in situ hybridization assay for detection of MCV genome in clinical specimens. Human fetal diploid lung cell monolayers were infected with clinical specimens, and after proper incubation and fixation in paraformaldehyde, hybridization was performed under full stringency conditions with a molecularly cloned biotinylated probe. Only MCV infected cells showed homology to the MCV probe with a purple-brown cytoplasmic staining. Additionally, we have described an in situ hybridization assay for direct detection of MCV genome in formalin-fixed, paraffin-embedded biopsies. Characteristic intracytoplasmic Molluscum bodies (Henderson-Paterson bodies) were detected in stratum spinosum cells of the epidermis. Striking staining similarities have been observed between in situ hybridization and haematoxylin-eosin cytostaining. These procedures are the first successful identification of MCV genome in clinical samples by molecular hybridization, with sensitivity and specificity equal to or greater than electron microscopy.

In situ hybridisation in perspective.

In the introduction to this review two questions were posed: is the technology associated with ISH ready for general use, and will the method become an important investigative tool? With the exception of the demonstration of some single and low copy sequences, non-radioactive ISH is now sufficiently developed and simplified to make it a routine technique. It is also clear that ISH will continue to have an important research role. In diagnostic pathology the technique is already providing valuable information and the present decade should see the development of many more diagnostic applications.

Automated immunochemistry.

A prototype immunostainer, featuring totally enclosed slide chambers, a reagent carousel and a microcomputer controlled fluid transfer system, was developed. The machine offers total flexibility in choice of primary and detection reagents for each slide and has been successfully used for the immunochemical demonstration of a variety of antigens in paraffin wax and blood smear preparations. It is considerably cheaper to use than manual immunochemistry and will be even cheaper in a production version currently under development.

Comparative quality assessment in immunocytochemistry: pilot study of CD15 staining in paraffin wax embedded tissue in Hodgkin's disease.

The feasibility of comparative quality assessment studies in immunocytochemistry was examined. The reactions of three CD15 antibodies--anti-Leu M1, DM1, and Tü9--were examined in paraffin wax sections in Hodgkin's disease under a variety of different fixation and pre-treatment conditions, using four immunochemical detection techniques. All three antibodies stained Reed-Sternberg cells, but DM1 could be used at slightly higher dilutions to achieve comparable results. Tissue fixed in formol sublimate showed the most intense staining reactions, and formol saline and neutral buffered formalin gave relatively poor results. Although neuraminidase pre-treatment improved staining, its routine use is probably contraindicated by its high cost. Trypsinisation has some value for sections of tissue fixed in formol saline and neutral buffered formalin. The avidin-biotin complex technique produced the best results, but indirect immunoperoxidase produced acceptable results, is technically easier to perform, and is less expensive. It is concluded that information regarding variations in techniques and commercially available reagents, which may be of use in routine diagnostic histopathology, can be obtained by comparative quality assessment studies of this type.

Multisite evaluation of a monoclonal antibody reagent (Syva) for rapid diagnosis of cytomegalovirus in the shell vial assay.

A pre-cytopathic effect (CPE) monoclonal antibody reagent (Syva Co., Palo Alto, Calif.) was evaluated in four laboratories for the rapid detection of cytomegalovirus (CMV) in shell vial cell cultures at 16 to 24 h and 40 to 48 h postinoculation. Results were compared with those obtained by inoculation of the specimen into conventional tube cell cultures that were examined for the presence of typical CMV CPE and subsequently tested by reaction with the monoclonal antibody reagent in an indirect immunofluorescence test. Of 937 specimens, CMV was positive in 184 (20%). CMV was detected twice as frequently in shell vials only (n = 29) as in conventional tube cell cultures (n = 14). Pre-CPE shell vial assay was 91% sensitive (range, 84 to 98%) and 96% specific (range, 93 to 98%) compared with the detection of CPE in conventional tube cell cultures. Overall, 137 of 166 (83%) and 143 of 166 (86%) of the CMV strains were detected at 16 to 24 h and 40 to 48 h postinoculation, respectively. The Syva reagent produced sensitive and specific results for the rapid detection of CMV infection in shell vial cell cultures and reliably confirmed the presence of the virus as detected by CPE in conventional tube cell cultures.

Amplification techniques for detection of herpes simplex virus in neonatal and maternal genital specimens obtained at delivery.

Viral culture amplification methods, including centrifugation, use of a cell pretreatment medium containing dimethyl sulfoxide and dexamethasone (DEX medium), and three commercial enzyme immunoassays (EIAs), were evaluated for use with low-titer, asymptomatic neonatal and obstetric samples obtained at delivery. The 4.5-h Ortho EIA was 59.7% sensitive at 16 h compared with 86.4% at 36 h in a spin-amplified format (SAT-EIA). After 36 h with DEX medium, the SAT-EIA was 97.3% sensitive using the 2.5-h modified Ortho EIA and 69.9% sensitive using the Fairleigh Dickinson EIA. Herpes simplex virus (HSV) cytopathic effect was reported in tube culture 1 to 2 days earlier in 8.1 to 35.9% of cells with DEX medium. Overall, the SAT-EIA using the modified Ortho EIA and cells with DEX medium detected 147 of 153 (96.1%) HSV isolates from samples obtained predelivery and 93.1% of 29 HSV positives from samples obtained at labor and delivery, with specificities of 100 and 99.9%, respectively, at 36 h.

Southern blot analysis of DNA extracted from formal-saline fixed and paraffin wax embedded tissue.

Model experiments were designed to assess whether DNA could be recovered from formol-saline fixed peripheral blood lymphocytes and tonsil tissue for use in Southern blot gene analysis. Lymphocytes were fixed for 30 min and tonsil for 6 and 24 h, then paraffin embedded. High molecular weight DNA was extracted by prolonged digestion (2-7 days) with proteinase K or protease XXIV in the presence of 1 per cent sodium dodecyl sulphate. Restriction, transfer and hydridization were possible without modification of standard procedures. Multiple copy sequences were demonstrated using Mspl and Bst Nl restriction and hybridization for the Y chromosome (pHY 2.1 probe), single copy genes using EcoRI and BamHl restriction for the T-cell receptor beta chain (T beta probe), and Bgl II and Hind III for the immunoglobulin heavy chain (JH probe). Identical banding to unfixed tissue was achieved except when 24 h fixed extracts were used. With these, demonstration of the 24 KB Bam Hl/T beta and 9.2 KB Hind III/JH bands was not obtained. These findings suggest that as the fixation time is extended, alterations to DNA will limit the available range of restriction enzyme/probe combinations. However, with careful choice of these the extraction of DNA from formalin fixed and paraffin embedded pathological tissue for Southern blotting should be profitable.

Evaluation of a commercial monoclonal antibody for detection of adenovirus antigen.

The Adenoclone monoclonal antibody for detection of adenovirus antigen was evaluated by enzyme immunoassay and a 72-h indirect fluorescent-antibody test in shell vials and compared with standard culture. The sensitivity and specificity of the Adenoclone test were both 100% with 114 cell culture isolates. With 310 direct clinical specimens, the Adenoclone enzyme immunoassay sensitivity varied from 14.3 to 66.6%, but the Adenoclone indirect fluorescent-antibody test sensitivity was 86.7%, with 100% specificity.