Parvovirus b19 DNA is commonly harboured in human skin.

BACKGROUND: Parvovirus B19 is the aetiological agent of erythema infectiosum. The presence of B19 DNA in lesional skin of other cutaneous manifestations has frequently been reported although there is disagreement on the role of the B19 virus in tissues. OBJECTIVES: To investigate the presence of B19 DNA (1) in skin lesions of patients with a described B19-related disease, (2) in skin lesions of B19-unrelated diseases and (3) in healthy skin. METHODS: A total of 121 skin samples were examined for the presence of B19 DNA by PCR assays and peptide-nucleic-acid-based in situ hybridisation techniques. RESULTS: B19 DNA was detected in 11/38 (28.9%) pityriasis lichenoides, 8/30 (26.7%) melanocytic naevi, 5/29 (17.2%) primary melanomas and 6/24 (25.0%) healthy skin biopsies. A difference in B19 DNA prevalence was observed in specimens grouped according to age, irrespective of pathologies. CONCLUSIONS: B19 DNA can be found in skin tissues of patients with pityriasis lichenoides as well as in lesions not related to B19 infection and in healthy controls. B19 DNA can be detected in skin of young subjects in a significantly high rate compared to adults, suggesting that viral persistence may be the usual outcome after primary infection.

Diagnosis of fetal parvovirus B19 infection: value of virological assays in fetal specimens.

OBJECTIVE: The purpose of our work was to examine the most reliable laboratory diagnosis of fetal parvovirus B19 infection in hydropic fetuses by evaluating the most appropriate clinical sample and laboratory test. DESIGN: B19 DNA detection in fetal samples and serological signs of B19 infection in the respective mothers. Samples collected between January 2000 and July 2008. SETTING: Microbiology, University of Bologna, Bologna, Italy. SAMPLES: One hundred thirty-five fetal samples (58 fetal cord blood and 77 amniotic fluid samples) and 109 serum samples collected from 109 pregnant women. METHODS: Validated and certified in situ hybridisation assay (ISH) and polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA) were performed on fetal samples to detect B19 DNA. B19-specific antibodies were investigated in maternal serum samples by a commercial enzyme immunoassay. MAIN OUTCOME MEASURES: Parvovirus B19 DNA detection in fetal specimens was analysed in relation to maternal serological signs of infection. RESULTS: Parvovirus B19 DNA was detected in 22.41% of fetal cord blood and 36.36% of amniotic fluid samples. A statistically significant difference was found between DNA detection by ISH (23.70%) and PCR-ELISA (14.81%) (P= 0.004). Only 11.76% of fetuses with virological diagnosis of B19 infection were from women with serological signs of acute/recent B19 infection. CONCLUSIONS: Diagnosis of fetal parvovirus B19 infection cannot always rely on maternal serological investigations but rather on the virological analysis of fetal samples. Both fetal cord blood and amniotic fluid samples are suitable for diagnosis, but the detection of B19 DNA in the cells of amniotic fluid samples by ISH proved to be the most reliable diagnostic system.

Seroprevalence of IgG against conformational and linear capsid antigens of parvovirus B19 in Italian blood donors.

Serum samples from 446 Italian blood donors between 18 and 65 years of age were analysed for the presence of IgG against parvovirus B19 capsid proteins VP1 and VP2 including conformational and linear epitopes. The overall prevalence of IgG against parvovirus B19 capsid proteins VP1 and VP2 against at least one antigen type was 79.1 %. No significant difference was found between men and women. In the 18-27 years age group, 77.0 % of the population had experienced infection with the virus, reaching 88.5 % in the 48-57 years age group. The overall prevalence of IgG was 78.0 % against conformational VP1 + VP2 antigens, 74.9 % against conformational VP2, 70.9 % against linear VP1 and 23.3 % against linear VP2 in the analysis of the IgG response against different conformational and linear epitopes of VP1 and VP2. Although IgG against conformational VP1+VP2, conformational VP2 and linear VP1 was present in more than 60 % of subjects in all age groups, IgG against VP2 linear antigens was present in only 32% of subjects in the 18-27 years age group and then decreased to 20.5 % in the 28-37 years age group. A different trend was noted when IgG positivity against linear and conformational epitopes was analysed separately in men and women. A significant increase was found in seroprevalence of IgG against VP2 conformational antigens with increasing age in males and a significant decrease in seroprevalence of IgG against VP2 linear antigens in women with increasing age.

Antibody response to B19 parvovirus VP1 and VP2 linear epitopes in patients with haemophilic arthritis.

Parvovirus B19 infection occurs very frequently in patients with haemophilia on account of its transmission with plasma derivatives. In order to achieve a more defined serological pattern for the study of the role of B19 infection in haemophilic arthritis, 53 serum samples from 37 patients with haemophilic arthritis were investigated for the presence of IgG immune response against B19 VP2 and VP1 linear epitopes and VP2 conformational antigen compared to the serological reactivity against B19 NS1 and to the presence of B19 DNA in the synovial membranes. An IgG immune response against VP1 and VP2 linear epitopes was detected by immunoblot assay using recombinant proteins expressed in Escherichia coli. Specific IgG against VP2 and VP1 linear epitopes were present in 84.90 and 92.45% of haemophilic arthritis patients and in 28.0 and 64.0% of the controls (P<0.001) respectively. All 53 sera of the haemophiliacs (100%) and 66.0% of the controls (P<0.001) were IgG positive and IgM negative against VP2 structural epitopes. Specific IgG against VP2 linear epitopes, which are a serological marker of active or very recent B19 infection, proved to be significantly associated with the presence of anti-NS1 antibodies and with the presence of B19 DNA in synovial tissue in patients with haemophilic arthritis. In conclusion, in these patients the presence of B19 IgG anti-VP2 linear epitopes, in absence of IgM anti-VP2 structural antigens, can be a useful serological marker to diagnose active, recent or persistent B19 infection.

Detection of parvovirus B19 IgG: choice of antigens and serological tests.

Serum samples were analysed for the presence of (a) IgG against VP1+VP2 using recombinant native conformational antigens by ELISA test (b) IgG against VP2 using recombinant native conformational antigens by ELISA test and (c) IgG against VP1 and against VP2 using denatured linear antigens by Western blot. Out of the 446 samples examined, 353 were positive for specific B19 IgG and out of these, 98.6 % proved positive in the ELISA assay using conformational VP1+VP2 antigens, 94.6% proved positive in the ELISA assay using conformational VP2 antigens, 89.5% were positive at the Western blot assay using denatured linear VP1 and VP2 antigens, with all proving positive for linear VP1 and only 29.5% out of the positive samples proving positive for linear VP2. Since all samples positive by Western blot proved positive by ELISA, our data show that recombinant capsids obtained either with VP1+VP2 or with VP2 alone, used in ELISA, are very useful for detecting the immune response against both conformational and native linear epitopes of B19 structural proteins although some sera may have antibodies directed exclusively against VP1+VP2 antigens and few may have antibodies directed exclusively against VP2 antigens alone.

Relevance of B19 markers in serum samples for a diagnosis of parvovirus B19-correlated diseases.

In order to evaluate the optimal and essential diagnostic test(s) for a correct diagnosis of B19 diseases, 344 consecutive serum samples were tested from 344 patients with clinical suspicion of B19 infection during an epidemic period (early Spring-Autumn 2000). Sera were tested for B19 DNA by a standardized competitive polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA) and dot-blot hybridization and for specific IgM and IgG by ELISA. Of 344 patients examined, 125 were positive for markers of B19-associated disease: 49 had both B19 DNA and IgM, 50 had B19 DNA without IgM, and 26 had IgM without B19 DNA. After examination of the different patterns of B19 markers as diagnostic tools for B19 infection, IgM determination detected only 60% of B19-documented infections. IgM tests were nevertheless fundamental, as they were the unique diagnostic marker in 20.8% of documented infections (26 of 125 patients), in the diagnosis of recent, but still symptomatic infections when B19 DNA was no longer detectable. The determination of B19 DNA with PCR permitted detection of 79.2% of infections and therefore represented an essential test. PCR was fundamental for the diagnosis of B19 disease, as the unique diagnostic marker in 32% of documented infections (50 of 125 patients), both in acute infections at the onset of symptoms before the appearance of immunological response, and during the course of persistent B19 infections in which IgM had cleared. The contemporaneous determination of B19 DNA by PCR and specific IgM appears to be the most appropriate diagnostic protocol for the correct laboratory diagnosis of B19 infection.

Identification of an immunodominant peptide in the parvovirus B19 VP1 unique region able to elicit a long-lasting immune response in humans.

The immune response against parvovirus B19 is mainly directed against the two structural proteins, VP1 and VP2. The amino terminal half of the VP1 unique region has been shown to elicit a dominant immune response in humans, more effective than other linear epitopes and also it has been seen to contain significant neutralizing linear epitopes. Three overlapping recombinant peptides corresponding to amino acids 2-40 (VP1-A), amino acids 32-71 (VP1-B), and amino acids 60-100 (VP1-C) of the VP1 unique region were produced by a procaryotic expression system. These peptides were used as antigens in a Western blot assay to detect specific immunoglobulin G (IgG) in serum samples from blood donors of different age groups with documented signs of a past B19 infection. Fragment VP1-C appeared significantly immunodominant over the other peptides, reacting with specific IgG in 86% of serum samples. The fragment VP1-C corresponds to a sequence with a known neutralizing activity and seems able to elicit a long-lasting immune response because specific IgG were present in blood donors of all age groups. VP1-C would therefore appear to be an attractive candidate as a component of a subunit vaccine.

Differential IgM response to conformational and linear epitopes of parvovirus B19 VP1 and VP2 structural proteins.

The IgM immune response against conformational and linear epitopes of B19 structural proteins VP1 and VP2 was examined in serum samples with a suspect B19 infection to determine the most suitable antigen for use in IgM detection and also to evaluate a possible relationship between the course of B19 infection and the presence of epitope type-specific IgM. The detection of IgM against conformational epitopes was performed by ELISA using undenatured VP1 and VP2 antigens whereas the detection of IgM against linear epitopes was performed by Western blot assays using denatured VP1 and VP2. IgM immune response against VP1 conformational epitopes appeared dominant, being detected in all serum samples positive for specific IgM, whereas IgM against VP2 linear antigen were found less frequently, being identified in less than half of the B19 IgM positive sera. In the examination of the course of infection, IgM against VP1 conformational epitopes appeared in the active phase of B19 infection at the same time and with the same frequency as IgM anti VP2 conformational epitopes and anti linear VP1 epitopes. IgM against VP1 conformational epitopes were seen to be long-lasting because in the recent phase of infection they were still present when other specific IgM were absent. During the active phase of B19 infection, IgM against VP2 linear epitopes were less frequently found than other specific IgM and in the recent phase they underwent a rapid temporal diminution. The data demonstrate that a sensitive B19 IgM test needs to be performed in diagnostic laboratories by ELISA using conformational B19 antigens; Western blot assays can be used only as confirmatory tests using VP1 linear antigens.

A system to enhance the sensitivity of digoxigenin-labelled probe: detection of B19 DNA in serum samples.

A highly sensitive dot-blot hybridisation assay for the routine screening of numerous samples is described, using parvovirus B19 as a model. Digoxigenin-labelled B19 DNA probe was constructed by PCR, hybrids were detected by an anti-digoxigenin monoclonal antibody followed by a second step, using anti-mouse antibodies conjugated to an alkaline phosphatase-dextran complex (EnVision, Dako) was carried out. The sensitivity of the assay was evaluated using both colourimetric and chemiluminescent substrates for the alkaline phosphatase and was compared with a dot-blot hybridisation assay using the digoxigenin-labelled probe and a standard detection system. With the colourimetric substrate, the EnVision system was able to detect 10 fg of B19 DNA, while with the chemiluminescent substrate the sensitivity increased by up to 2 fg (6 x 10(2) genome copies). This detection system was shown to increase the sensitivity of the assay compared to the standard colourimetric visualisation for the digoxigenin-labelled probe, which could detect 0.1 pg. On account of its sensitivity and specificity the dot-blot hybridisation assay together with the chemiluminescent substrate for the EnVision detection system was used to analyse 760 serum samples; the same sera were tested for B19 DNA with the standard colourimetric visualisation for the digoxigenin-labelled probe used routinely in the diagnostic laboratory.

Different patterns of restriction to B19 parvovirus replication in human blast cell lines.

B19 parvovirus can replicate in erythroid progenitor cells and in a small number of human blast cell lines. To better understand and analyze the B19 virus replicative cycle, we performed and compared the infection of bone marrow cells and of different blast cell lines with erythroblastoid and megakaryoblastoid phenotypic characteristics (UT-7, TF-1, M-07, and B1647). Following in vitro infection, B19-specific nucleic acids were characterized with regard to the genome-replicative intermediates, the transcription pattern, and the localization of virus-specific nucleic acids inside infected cells. While all cell lines tested proved to be susceptible to B19 virus infection, two different patterns of restriction to replication of B19 virus were observed. In the first restriction pattern, observed in UT-7 cells, the single-stranded viral DNA was converted to double-stranded replicative intermediates, identical to those found in bone marrow cells, and a full set of viral transcripts were observed. However, replication and transcription were restricted to a small subset of cells, and production of capsid proteins was not detected. In the second restriction pattern, observed in TF-1, M-07, and B1647 cells, the single-stranded viral DNA was not converted to double-stranded replicative intermediates.

Immunoreactivity against linear epitopes of parvovirus B19 structural proteins. Immunodominance of the amino-terminal half of the unique region of VP1.

Three peptides corresponding to the 2-100 amino acids of VP1 unique sequence (VP1-F1), to the 99-227 amino acids of VP1 unique sequence (VP1-F2) and to the 237-781 amino acids of VP1 protein common to VP2 (VP1-F3 = VP2) were produced by prokaryotic expression. The three peptides, which span the entire VP1 structural protein of parvovirus B19 and also the entire VP2 protein, were used to evaluate the immunoreactivity against linear epitopes of these fragments in a large number of serum samples taken in different clinical situations with regards to B19 infection and in some commercial preparations of aspecific immunoglobulins. The data demonstrated that the specific VP1-F1 fragment, corresponding to the amino-terminal half of the VP1 unique region, is immunodominant and can elicit a long lasting immune response in comparison with VP1-F2 and VP1-F3 = VP2. Data regarding the presence of specific IgG to the three fragments in commercial preparations of immunoglobulins demonstrated that the dominant immune response was also against VP1-F1 linear epitopes while IgG against VP1-F2 and IgG against VP1-F3 = VP2 could be found only in high concentrations of Ig preparations. The reported data can be useful as a basis for the development of a B19 recombinant vaccine.

Occurrence and clinical role of active parvovirus B19 infection in transplant recipients.

To evaluate the occurrence and clinical role of active parvovirus B19 infection in solid organ and bone marrow transplant recipients, 256 serum samples from 212 transplant patients were investigated retrospectively by competitive polymerase chain reaction. Sera were drawn during the transplantation period and up to 6 months after transplantation during a nonepidemic 1-year period. Three patients were found positive for B19 DNA; only one liver transplant patient had a clinically overt B19 infection. Overall, the rate of active parvovirus B19 infection in transplant subjects was low (1.42%), probably due to the high number of actively or passively immunized subjects among transplant recipients; this may also account for the asymptomatic infections observed.

Chemiluminescence Western blot assay for the detection of immunity against parvovirus B19 VP1 and VP2 linear epitopes using a videocamera based luminograph.

A chemiluminescent Western blot (WB) assay was developed to detect the immune status against linear epitopes of parvovirus B19 structural proteins VP1 and VP2. The chemiluminescent WB assay combined the sensitivity of chemiluminescent substrates and the objective evaluation of the luminescent signal obtained with a new system which consists of a videocamera-based, high-performance, low light level imaging luminograph connected to a personal computer for image analysis. The potential for diagnostic purposes was evaluated using reference serum samples and 75 clinical samples from patients with different clinical conditions and laboratory evaluations regarding B19 infection. The chemiluminescent Western blot assay provided reproducible results, an objective evaluation of the results and a semi-quantitative analysis of the presence of antibodies against VP1 and VP2 in human sera. The chemiluminescent Western blot assay proved more sensitive than the classic colourimetric Western blot assay.

Standardization of a PCR-ELISA in serum samples: diagnosis of active parvovirus B19 infection.

To standardize a PCR assay for the detection of parvovirus B19 DNA in serum samples three different sample treatments were evaluated on the basis of the efficiency of recovery, reproducibility, convenience of sample handling, and presence of PCR inhibitors. Moreover, the presence of an internal standard competitor as the working reagent at one defined concentration in a competitive PCR-ELISA has been suggested as a valid tool to standardize and validate the assay. The results indicated that serum sample treatment by rapid heating fulfilled the criteria for a routine practice in the diagnostic laboratory. Titration experiments carried out to define the optimal amount of the internal standard competitor to use in PCR-ELISA showed that at 2 x10(2) competitor copies, any amplification interferences between target and competitor sequences were avoided. The internal standard competitor in a competitive PCR-ELISA allows the detection of false-negative results due to PCR inhibitors in the samples or large amounts of target DNA. Heating treatment and competitive PCR-ELISA for the detection of parvovirus B19 DNA were applied to the testing of 347 serum samples, which were submitted to the laboratory for B19 investigation. Of the 34 serum samples that were positive for B19 DNA, 15 were from adult patients and 19 from pediatric subjects. B19 infection was associated with haematological disorders, nonimmunological foetal hydrops, atypical rash, arthropathies, hepatic dysfunction, nonspecific symptoms, and congenital infections.

Prenatal diagnosis of parvovirus B19-induced hydrops fetalis by chemiluminescence in situ hybridization.

Parvovirus B19 can be transmitted transplacentally from the infected mother to the fetus during pregnancy, and hydrops fetalis, abortion, or stillbirth can result. In our study we explored the use of chemiluminescence in situ hybridization to detect B19 DNA on cord blood cells, amniotic fluid cells, and pleuric fluid cells from several cases of hydrops fetalis. B19 DNA was detected by using digoxigenin-labeled probes immunoenzymatically visualized with the chemiluminescent adamantil-1,2-dioxetane phenyl phosphate substrate for alkaline phosphatase. The luminescent signal emitted from the hybridized probes was detected, analyzed, and measured with a high-performance, low-light-level imaging luminograph connected to an optical microscope and to a personal computer for the quantification and localization of the chemiluminescent emission inside individual cells.

IgG immune response to B19 parvovirus VP1 and VP2 linear epitopes by immunoblot assay.

Human B19 parvovirus recombinant capsid proteins VP1 and VP2 were expressed in E. coli and purified. Recombinant proteins were used to detect a specific IgG immune response against VP1 and VP2 linear epitopes by immunoblot assay. A total of 222 serum samples from 218 apparently immunocompetent subjects with different clinical conditions and laboratory evaluations with regards to B19 infection were analyzed. The sera had previously been tested for B19 DNA and for specific IgM and IgG against VP2 conformational antigens by ELISA assay. The data show that, during the active or very recent phase of infection, IgG anti-VP1 linear epitopes appear in concomitance and with the same frequency as IgG anti-VP2 conformational antigens. IgG against conformational VP2 antigens and against linear VP1 epitopes seem to persist for months or years in the majority of individuals. IgG against VP2 linear epitopes are generally present during the active or very recent phase of infection and during the convalescent phase, while they are present only in about 20% of subjects with signs of a past B19 infection.

IgG response to the immunoreactive region of parvovirus B19 nonstructural protein by immunoblot assay with a recombinant antigen.

A total of 190 serum samples from patients with different clinical manifestations in whom B19 parvovirus infection was suspected and 108 control serum samples were analyzed for the IgG response against parvovirus B19 nonstructural protein. An immunoblot assay was developed using the immunoreactive region of the B19 nonstructural protein as recombinant antigen. Virologic (B19 DNA) and serologic (IgM and IgG anti-VP) markers of B19 infection were also investigated. There was an even distribution of IgG nonstructural antibodies in serum samples of parvovirus B19-infected patients and controls (27.7% and 21.7%, respectively) that were mainly associated with patterns of past B19 infection.

Evaluation of immunoassays for the detection and typing of PCR amplified human papillomavirus DNA.

AIMS: To evaluate different hybridisation techniques to detect and type human papillomavirus (HPV) DNAs amplified by consensus primer polymerase chain reaction (PCR) in biopsy and cytological specimens. METHODS: A hybrid capture-immunoassay in microtitre wells was performed to detect HPV sequences amplified by PCR and typed by specific oligoprobes. Consensus primers were used to amplify a sequence within the L1 open reading frame, and direct digoxigenin labelling of amplified products was performed during the amplification reaction. The amplified product was separately hybridised with six biotinylated type specific probes (HPV6, 11, 16, 18, 31, and 33); hybrids were then captured into streptavidin coated microtitre wells and detected by a spectrophotometer as an ELISA using antidigoxigenin Fab fragment labelled with peroxidase and a colorimetric substrate. The results were compared with the dot-blot immunoassay used to detect and type PCR amplified HPV DNA sequences. Consensus primers were used to generate the same unlabelled PCR product; digoxigenin labelled type specific probes for HPV6, 11, 16, 18, 31, and 33 were used and hybrids visualised by colorimetric immunoenzymatic reaction. Thirty nine biopsy specimens and 31 cytological samples were tested by the PCR-ELISA and by standard PCR followed by dot-blot hybridisation. RESULTS: The PCR-ELISA proved to be more sensitive than standard PCR with dot-blot hybridisation typing. All samples positive for HPV-DNA in standard PCR with dot-blot hybridisation method were confirmed positive by the PCR-ELISA assay; however, seven samples were positive only by PCR-ELISA. CONCLUSIONS: The PCR-ELISA assay, which can be performed in one day, is easily standardised and therefore seems to be a practical, sensitive, and reliable diagnostic tool for the detection and typing of HPV genomes in biopsy and in cytological specimens in the routine diagnostic laboratory.

B19 parvovirus induced fetal hydrops: rapid and simple diagnosis by detection of B19 antigens in amniotic fluids.

In our study we describe the direct detection of parvovirus B19 capsid antigens in amniotic fluid samples for the rapid and simple prenatal diagnosis of B19 induced fetal hydrops. The assay was performed on amniotic fluid specimens from fetal hydrops dotted on nylon membranes. The two capsid antigens, VP1 and VP2, which represent four per cent and 96 per cent of the capsid, respectively, were detected using a pool of monoclonal antibodies directed against these two proteins and the complex was visualized by immunoperoxidase staining. The assay could be performed in about four hours and positive results were revealed at the end of the reaction as dark blue spots on the nylon membrane. We analysed 26 amniotic fluid samples from 26 selected cases of non-immune hydrops for the presence of B19 antigens. Out of these 26 samples, 13 had previously proved positive for B19 DNA, detected by dot blot hybridization and/or in situ hybridization and/or nested PCR, and 13 had proved negative. The results obtained with our assay were compared with results obtained for the presence of B19 DNA and a close agreement was found. The method is simple and rapid to perform, does not require costly instruments, and all the reagents used in the assay are commercially available. The assay described can thus be useful for a prompt counselling and management of B19 fetal infection.

Chemiluminescence: a sensitive detection system in in situ hybridization.

Chemiluminescence is the light emission produced by a chemical reaction in which chemically excited molecules decay to the ground state. The phenomenon is utilized in various analytical techniques in which small amounts of analytes or enzymes can be detected and quantified by measurement of the light emitted by bio- or chemiluminescent reactions. Recently chemiluminescence has been proposed as a valid alternative to radioactive or colorimetric methods in in situ hybridization assays, in which target nucleic acids are localized by labeled probes inside individual cells with the preservation of cell morphology. Chemiluminescence in situ hybridization is performed using probes that are detected using enzymes with their appropriate chemiluminescent substrates. The luminescent signal from the hybrid formation is detected, analysed and measured with a high performance low light level imaging apparatus connected to an optical microscope and to a personal computer for quantitative image analysis. Generally, the instrumental system to detect positive signals after in situ hybridization operates in three steps: firstly tissue structures and cells are recorded in transmitted light then the luminescent signal is measured with an optimized photon accumulation; and then, after a computer elaboration of the luminescent signal with pseudocolors corresponding to the light intensity, an overlay of the two images on the screen provided by the transmitted light and by the luminescent signal allows the spatial distribution of the labeled probe to be localized and evaluated. The main advantages of chemiluminescence in situ hybridization are mainly the sensitivity, the quantification of the data, the objectivity of the evaluation and the digital imaging of the results. The chemiluminescence in situ hybridization assay, which can be applied to cell smears, archival frozen and paraffin embedded tissue samples, can be a useful tool for a sensitive and specific diagnosis of viral infections and for the detection and study of specific genic sequences inside the cells. The use of the chemiluminescent in situ hybridization assay is also promising for an estimation and quantification of nucleic acids present in tissue samples or cellular smears and for imaging gene expression in cells.