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AIMS Because serous cystadenoma (SCA) does not usually require excision, it is critical to distinguish it from differential diagnoses which do, especially neuroendocrine tumour (NET). The gold standard for diagnosing SCA is assessment of endoscopic ultrasound-guided fine needle aspiration/biopsy (EUS-FNAB) material. Inhibin immunohistochemistry aids this assessment, but such positivity is not absolutely sensitive or specific to SCA. The following is the largest known study of SCA EUS-FNAB specimens and the first to compare four potential SCA immunomarkers between themselves and inhibin, compared against NET. METHODS AND RESULTS Immunohistochemistry for calponin, mucin 6 (MUC6), glucose transporter 1 (GLUT1) and vascular endothelial growth factor A (VEGFA) was performed on 30 EUS-FNAB and three resection specimens of SCA and 32 EUS-FNAB specimens of NET. GLUT1 and VEGFA were suboptimal as diagnostic immunomarkers of SCA, being expressed by 10 and 44% of NETs, respectively. Further, their expression by cellular constituents of blood which often contaminate EUS-FNAB specimens hampered identification of neoplastic cells, especially in hypocellular samples. While 19% of NETs showed nuclear MUC6 positivity, cytoplasmic expression of the protein showed 100% specificity and sensitivity as an SCA marker. However, assessing MUC6 in EUS-FNAB specimens must also consider the protein's focal expression in physiological pancreatic, gastric or duodenal tissues, which can contaminate these specimens. Calponin was less sensitive (71% versus 100%) but more specific (100% versus 91%) than inhibin, although easier to assess in EUS-FNAB specimens than MUC6. CONCLUSIONS Of the four potential immunomarkers of SCA suggested by the existing literature, calponin and MUC6 are useful complementary studies to inhibin for application to EUS-FNAB specimens.

The relative distribution of lymphocyte subpopulations in the blood and liver of patients with primary sclerosing cholangitis (PSC) and related diseases has been studied using immunoenzyme techniques. The peripheral blood CD4/CD8 T lymphocyte ratio was significantly higher in active ulcerative colitis (UC) and in PSC with inactive UC than in inactive UC alone. In contrast, no relationship with disease activity was seen in Crohn's disease. The portal tract t lymphocyte count per high power field (mean +/- S.D.) was higher in pre-cirrhotic PSC (173 +/- 105) and primary biliary cirrhosis (PBC: 210 +/- 110) than in histologically normal liver (42 +/- 27). However, the overall portal tract CD4/CD8 ratio was similar in PSC (1.49), PBC (1.89) and normal controls (1.63). The results are consistent with immunological involvement in the pathogenesis of PSC, but argue against the hypothesis that changes in the peripheral blood T cell subsets are due to sequestration at the site of tissue inflammation.

Several studies have suggested that p53 immunostaining does not occur in benign mesothelium but is common in malignancies involving the serous surfaces, including malignant mesothelioma. As a result, p53 has been advocated as a marker of malignancy in serous fluid cytology. However, the specificity of p53 in this context has been brought into question by some studies that claim to have found immunostaining in benign mesothelium. The aim of our study was to examine p53 immunostaining in a large series of serous fluids to try to resolve the uncertainty. Monoclonal Do-7 antibody was used to immunostain ethanol-fixed cytospin preparations employing an alkaline phosphatase method. Positivity was found in 17 of 35 malignant effusions, including two probable mesotheliomas, but was not found in any of 115 benign effusions. Our study suggests that p53 immunostaining is a highly specific and moderately sensitive marker of malignancy in serous fluids.

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures (1). To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion (2). Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA (3). DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20°C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization (4) (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

Delta hepatitis (HDV) infection can only occur in the presence of hepatitis B (HBV) infection, as HDV requires a coat of HBV surface antigen (HBsAg) for assembly of complete virus. A number of studies have examined the variation of HBV markers in serum and liver during establishment of HDV infection, but none has systematically examined the relationship between the two viruses in individual hepatocytes. Liver biopsies from five patients with HDV/HBV infection were stained for HBsAg, HBV core antigen (HBcAg) and hepatitis D (delta) antigen (HDAg). Double immunostaining was performed with a combination of indirect immunoperoxidase and alkaline phosphatase/anti-alkaline phosphatase techniques. HDV and HBV antigens were expressed in all five liver biopsies. Co-localization of HBsAg was seen in up to 39% of HDAg positive cells, and HBcAg in up to 8% of HDAg positive cells. HBcAg was detectable in approximately 9% of HBsAg positive cells, and HBsAg in approximately 12% of HBcAg positive cells. HDV can replicate without HBV but ultimately requires HBV to produce complete virus and subsequently infect other cells. In this study the majority of HDV positive cells did not appear to contain HBV markers. This might suggest delta virus replication without assembly, or possibly sequential production/assembly of the virus.

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease 1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest 5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5'mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) 8. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA 2, 4, 9-11.

Mannan-binding protein (MBP) is a Ca(2+)-dependent lectin which was first described in 1978 in rabbit liver, and subsequently in serum and liver tissue from humans and a range of animal species. MBP structurally resembles C1q, and may act both as a focus for complement activation on the surface of microorganisms and as an opsonin in its own right. Low serum levels of serum MBP have been described in a group of children known to suffer from severe recurrent infections. MBP has also been reported to behave as an acute phase reactant. This preliminary study has investigated the localisation of MBP in human tissues using material obtained both at post mortem and from diagnostic liver biopsies. Using the IgG fraction of rabbit anti-human MBP, immunoperoxidase staining showed no evidence of significant MBP in a wide range of normal tissues, including liver taken both at post mortem and needle biopsy. However, there was a significant degree of staining for MBP in liver biopsies showing a variety of different pathologies, in particular severely damaged alcoholic livers, and those harbouring metastatic tumour. Moderate degrees of staining were also seen in liver biopsies from patients suffering from chronic biliary disease. It is uncertain whether this localisation of MBP in abnormal liver is an acute phase response, or represents a more fundamental link with liver disease. This question could be the focus for future studies.

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1. Recent advances in this technology have enabled high resolution comparison of whole genomes for the identification of genetic alterations in cancer and other genetic diseases 2. The Sub-Megabase Resolution Tiling-set array (or SMRT) array is comprised of a set of approximately thirty thousand overlapping bacterial artificial chromosome (BAC) clones that span the human genome in ~100 kilobase pair (kb) segments 2. These BAC targets are individually synthesized and spotted in duplicate on a single glass slide 2-4. Array CGH is based on the principle of competitive hybridization. Sample and reference DNA are differentially labeled with Cyanine-3 and Cyanine-5 fluorescent dyes, and co-hybridized to the array. After an incubation period the unbound samples are washed from the slide and the array is imaged. A freely available custom software package called SeeGH (www.flintbox.ca) is used to process the large volume of data collected - a single experiment generates 53,892 data points. SeeGH visualizes the log2 signal intensity ratio between the 2 samples at each BAC target which is vertically aligned with chromosomal position 5,6. The SMRT array can detect alterations as small as 50 kb in size 7. The SMRT array can detect a variety of DNA rearrangement events including DNA gains, losses, amplifications and homozygous deletions. A unique advantage of the SMRT array is that one can use DNA isolated from formalin fixed paraffin embedded samples. When combined with the low input requirements of unamplified DNA (25-100ng) this allows profiling of precious samples such as those produced by microdissection 7,8. This is attributed to the large size of each BAC hybridization target that allows the binding of sufficient labeled samples to produce signals for detection. Another advantage of this platform is the tolerance of tissue heterogeneity, decreasing the need for tedious tissue microdissection 8. This video protocol is a step-by-step tutorial from labeling the input DNA through to signal acquisition for the whole genome tiling path SMRT array.

A new monoclonal antibody, JC70, raised against a membrane preparation from a spleen affected by hairy cell leukaemia, recognises a membrane bound glycoprotein identical with that of the CD31 group of monoclonal antibodies. The antibody stains a fixation resistant epitope on endothelial cells in benign and malignant conditions in a wide variety of paraffin wax embedded tissue. JC70 stained malignant endothelial cells in 10 angiosarcomas with more consistency than monoclonal or polyclonal antibodies to factor VIII related antigen (FVIII-Rag). In four cases of Kaposi's sarcoma the antibody stained malignant endothelial cells but not spindle cells. It is concluded that antibody JC70 is of value for studying benign and malignant human vascular disorders in routinely processed tissue.

Identification of the renal erythropoietin-producing cells using transgenic mice. Regulation of erythropoietin production by the kidneys is central to the control of erythropoiesis. Uncertainty about the identity of the renal cells involved has been a major obstacle to understanding this mechanism. We have used sequence from the mouse erythropoietin locus to direct expression of a marker gene, SV40 T antigen, to these cells in transgenic mice. The transgenic constructs contained an oligonucleotide marker (Epo-M) or SV40 sequence (Epo-TAg) in the 5′ untranslated region of the mouse erythropoietin gene, flanked on each side by 9 and 7.5 kb of DNA from the mouse erythropoietin locus. Anemia-inducible expression of Epo-M and Epo-TAg was observed in the kidney. In one of thirteen lines, homologous integration of Epo-TAg into the mouse erythropoietin locus occurred. In transgenic mice bearing Epo-TAg at homologous and heterologous insertion sites, renal expression was restricted to a population of cells in the interstitium of the cortex and outer medulla. Immunohistochemical characterization by light and electron microscopy shows that these are the fibroblast-like type I interstitial cells.

The distribution of carbohydrate antigens of the ABO, Lewis, and Kell systems was examined in biliary and colonic epithelial of 11 patients with primary sclerosing cholangitis (PSC) using a panel of 11 monoclonal antibodies. Controls consisted of 27 liver biopsy specimens (11 normal, six alcoholic liver disease, five extrahepatic obstruction, and five primary biliary cirrhosis) and 24 colonic biopsy specimens (six normal, four Crohn's disease, and 14 ulcerative colitis). There was inappropriate staining with anti-A (four of six, 66%) and anti-B (nine of 11, 81%) in biliary epithelium of PSC patients compared with normal and disease controls. Expression of Lewis antigens was increased in patients with cholestatic liver disease. Ninety one per cent of PSC patients showed a similar pattern of inappropriate staining by anti-A and anti-B antibodies in colonic epithelium compared with 33% of normal and 42% of inflammatory bowel disease controls. There is inappropriate expression of A and B carbohydrate antigens in biliary and colonic epithelium in PSC. Whether these oncofetal antigens are implicated in the pathogenesis of this condition is discussed.

Late radiation damage to pig skin has been assessed at 104 weeks after exposure to sources of 90Sr/90Y (Emax 2.2 MeV) and 170Tm (Emax 0.9 MeV). Damage was assessed from measurements of dermal thickness in histological sections of irradiated skin and was compared with that of unirradiated skin to establish the relative reduction in dermal thickness. The size of the source varied from 0.1 to 40.0 mm in diameter; this covered the range of source sizes designed to simulate exposure to radioactive "hot" particles (or = 1.0 mm diameter) up to the lower range of field size that patients may be exposed to in radiotherapy treatments. Radiation doses were measured using an extrapolation chamber with a collecting electrode of 1.1 mm2, and thus the quoted doses represent an average dose over this area. For the larger 90Sr/90Y sources ofor = 5 mm diameter and the larger 170Tm sources ofor = 2 mm diameter there was no evidence, based on levels of damage consistent with aor = 10,or = 20,or = 30, andor = 40% reduction in relative dermal thickness, for any effect of source size on the ED50 value for each of these specified levels of effect. However, there was a marked effect of beta-particle energy; the skin surface doses associated with the ED50 values (+/- SE) for aor = 20% reduction in relative dermal thickness were approximately 12 and approximately 40 Gy for 90Sr/90Y and 170Tm, respectively. This difference in skin surface dose for an equivalent level of dermal injury reflects the variation in the depth dose from these two beta-particle emitters. These skin surface doses, for what was considered to be a clinically detectable dermal effect, were below the ED10 for the early skin response of moist desquamation. This supports the selection of late dermal thinning as the effect on which to base dose limits in radiation protection for more generalized larger area skin exposures. In comparison, single exposures to a small area, from sources designed to simulate those from hot particles, reinforced the view that acute ulceration should be the effect on which dose limitation is based. Either the isoeffect doses for a clinically detectable reduction in relative dermal thickness ofor = 20%, following a single exposure to a small area, were higher than the ED10 for acute ulceration or the area of skin showing dermal thinning was so small that it was not considered to be detrimental.

To assess the expression of p53 in premalignant lesions, we examined by immunohistochemistry benign colorectal adenomas (n = 72, measuring more than 6 mm and less than 95 mm in diameter) from patients without (group I, n = 23) or with (group II, n = 49) concurrent sporadic colorectal carcinomas. Using a panel of three monoclonal antibodies (PAb 240, PAb 421, PAb 1801) and two polyclonal antibodies (CM1, C19) immunohistological staining was demonstrated in 26% of the cases (19 of 72 adenomas, 7 of 23 from group I and 12 of 49 from group II). In the majority of the cases, p53 positive foci in the adenomas occurred in the most dysplastic areas, although focal positivity was detected in glands that were histologically normal. Expression of p53 protein was also detected in 21 of 30 (70%) colorectal carcinomas of group II. In two cases focal positive staining was observed in the polyps but not in the concurrent carcinomas. Non-neoplastic colonic mucosa and stromal lymphoid cells were negative in all cases examined. Over-expression of p53 in neoplastic tissues detected by immunocytochemistry is generally believed to correlate with the presence of mutation in the gene. This may not be an absolute rule, because in some hemopoietic malignancies, there is evidence that p53 protein may be detectable in the absence of an underlying mutation. These findings therefore represent the highest incidence in colorectal adenomas of abnormalities in the p53 protein expression, probably largely due to underlying mutations. This study also suggests that immunocytochemical demonstration of p53 protein may be a suitable method for the routine detection of subpopulations of cells which, by clonal expansion, could acquire a growth advantage within an adenoma during the neoplastic process.

AIMS: To test the hypothesis that fragments of platelet thrombi and vascular endothelium are incorporated into the walls of small cerebral vessels in systemic lupus erythematosus (SLE). METHODS: Six varied necropsy cases of central nervous system (CNS) SLE and 15 controls were studied. The controls were selected to represent a wide range of diseases in which the cerebral circulation is compromised. Tissue sections were stained by standard histochemical and immunocytochemical methods, the latter using antibodies to platelet membrane glycoprotein IIIa (CD61), and vascular endothelium (CD31). RESULTS: In four of six cases of CNS SLE characterised by small vessel hyalinization and thickening, fragments of platelet membrane were found in the walls of small cortical and meningeal vessels. Similar findings were not evident in two other SLE cases that were characterised by relatively short clinical histories and an acute vasculitis. One control case of severe polyarteritis nodosa showed platelet fragment deposition in arteries larger than the vessels so affected in SLE. CONCLUSIONS: Previous studies have suggested that neuropsychiatric symptoms in SLE may be related to repeated episodes of vasculitis in small cerebral vessels that are triggered by antiphospholipid antibodies. Concurrent thrombus formation might facilitate the incorporation of platelet fragments into small vessel walls. This process contributes to the thickening and irregularity of small vessels, a major feature of longstanding cases of CNS SLE.

OBJECTIVE: Our objective was to study the endothelial status of the luminal lining of uteroplacental vessels in the human placental bed in normal and abnormal pregnancy in the third trimester. STUDY DESIGN: Six placental basal plates from uncomplicated pregnancies and five from pregnancies complicated by preeclampsia ( n = 3), preeclampsia and a small-for-gestational-age infant ( n = 1), and diabetes mellitus ( n = 1 ) were accessioned from the archives because of documentation of their containing uteroplacental vessels. Five placental bed biopsy specimens with intraluminal endovascular trophoblast in the third trimester were also studied. Sections were subjected to immunohistochemical analysis with monoclonal and polyclonal antibodies labeling endothelium and trophoblast. RESULTS: In third-trimester normal uncomplicated pregnancies the uteroplacental arteries and veins were completely endothelialized with no disruption of the endothelium. In third-trimester abnormal pregnancies the uteroplacental veins were also completely endothelialized. However, intraluminal endovascular trophoblast was seen within the uteroplacental arteries in eight of the 10 complicated pregnancies; this finding was associated with disruption of the endothelium. CONCLUSION: In preeclampsia there is an aberrant wave of endovascular trophoblast migration in the third trimester, resulting in focal disruption of the endothelium. This may be responsible for the endothelial cell dysfunction thought to be of pathogenetic importance in preeclampsia.

Cytospin preparations were made from 102 serous effusions for immunocytochemical staining using a panel of monoclonal antibodies including a new monoclonal antibody Ber-EP4. On cytological examination, 32 fluids were reported to contain tumour cells consistent with metastatic adenocarcinoma; 66 contained benign cells only and three were reported to contain cells suspicious of malignancy. One eifusion contained tumour cells consistent with malignant mesothelioma. Positive staining of the tumour cells with Ber-EP4 was observed in the 32 effusions (100%) which contained adenocarcinoma cells. No staining of the mesothelial cells in these 32 specimens was observed. Carcinoembryonic antigen, epithelial membrane antigen Ca2 and CD 15 staining of tumour cells was noted in 53%, 50%, 50% and 9% of these cases, respectively. None of the mesothelial cells in the benign effusions stained with Ber-EP4. Nor did the malignant mesothelial cells in the only case of malignant mesothelioma. These findings suggest that Ber-EP4 is a valuable addition to antibodies available for the differential diagnosis of mesothelial cells and adenocarcinoma cells in serous effusions.

The L1 antigen was investigated as a marker of squamous differentiation in urothelium using a monoclonal antibody Mac387, and the results were compared with the expression of high molecular weight cytokeratins. L1 antigen was consistently demonstrated in all instances of partial and complete squamous metaplasia and in squamous carcinomas. In contrast, pure transitional cell carcinomas (except one with minor focal staining), adenocarcinomas and normal and hyperplastic urothelium did not label. In a few squamous carcinomas in situ, the pattern of labelling obtained with Mac387 was different from that seen in invasive squamous carcinomas and metaplasias. Compared with high molecular weight cytokeratins, the expression of L1 was more intense in areas of squamous differentiation. L1 expression, as identified by antibody Mac387, may therefore serve as a useful marker of squamous differentiation in urothelial lesions.

One hundred cases of routinely fixed and processed non-Hodgkin's lymphoma from Nigeria were immunostained with a small panel of monoclonal antibodies against B-, T- and macrophage antigens. The aims of the study were to assess the suitability of stored material from a country like Nigeria for immunohistochemical examination and the ability of the antibody panel to evaluate the distribution of B- and T-cell neoplasms. Eighty-seven of the 100 cases gave interpretable immunostaining, with 75 being B-cell and 12 T-cell neoplasms. Eighty-seven of the 100 cases gave interpretable immunostaining, with 75 being B-cell and 12 T-cell neoplasms. There were no tumours of macrophage lineage. Four cases gave satisfactory staining of reactive lymphoid cells but no reactivity with malignant cells and thus were not phenotyped. The remaining nine cases gave no staining of neoplastic or reactive cells, suggesting that they were unsuitable for immunohistochemical study, presumably because of inappropriate fixation and handling. We concluded that a panel of three monoclonal antibodies is suitable for routine immunostaining of conventionally fixed and processed blocks in Third World countries and will give diagnostically useful information in approximately 95% of cases.

Megakaryocytes in 63 bone marrow trephine biopsies were examined for their staining characteristics, location and size distribution using the monoclonal antibody Y2/51 directed against platelet glycoprotein IIIa (CD61). Megakaryocytes in normal bone marrow were evenly distributed and demonstrated homogeneous staining with Y2/51. In addition, there was little variation in their size or shape. In contrast, myelodysplastic and myeloproliferative bone marrow trephines showed considerable dysmegakaryopoiesis demonstrated by heterogeneity of staining, an altered architectural distribution with a predominantly paratrabecular location and considerable variation in size and shape. Furthermore, in myelodysplasia 25% of the CD61 positive cells were micromegakaryocytes as opposed to less than 10% in normal or reactive marrows. Such dysmegakaryopoiesis is believed to be a clinically important feature of myelodysplasia, although until now it has only been possible to assess it subjectively. The availability of the monoclonal antibody Y2/51 provides a rapid and reproducible means of studying megakaryocyte size, shape and distribution in routine trephine specimens and may help to overcome some of the diagnostic problems currently associated with myelodysplasia and other intrinsic bone marrow neoplasias.

In a series of nine angioleiomyomas, nerves were identified immunohistologically within the capsule and interstitium only in those that were painful. We believe that it is stretching of capsular nerves that underlies most of the patients symptoms.

Examines the background and reasoning behind building societies′ involvement in the commercial property lending sector. Explains how they are operating in the market. Looks at future developments, anticipating that building societies will continue to diversify.

A method for combined immunohistochemistry using alkaline phosphatase antialkaline phosphatase (APAAP) and in situ hybridisation using biotinylated probes was developed. The method requires no change to either technique and no additional procedures between them. The procedure was able to show the cell types involved in parvovirus infection of the fetus. The efficiency of immunohistochemistry and in situ hybridisation for the detection of cytomegalovirus in kidney were also compared: occasional cells contained cytomegalovirus DNA but not antigen. The method is rapid, straightforward, and has wide applications in the study of viral infections, genes, and gene products in tissue sections because it permits the combined demonstration of antigen and nucleic acid on the same section in routine clinical material.